Cunningham, Scott A

ABSTRACT Standardized approaches to phage susceptibility testing (PST) are essential to inform selection of phages for study in patients with bacterial infections. There is no reference standard for assessing bacterial susceptibility to phage. We compared agreement between PST performed at three centers: two centers using a liquid assay standardized between the sites with the third, a plaque assay. Four Pseudomonas aeruginosa phages: PaWRA01ø11 (EPa11), PaWRA01ø39 (EPa39), PaWRA02ø83 (EPa83), PaWRA02ø87 (EPa87), and a cocktail of all four phages were tested against 145 P . aeruginosa isolates. Comparisons were made within measurements at the two sites performing the liquid assay and between these two sites. Agreement was assessed based on coverage probability (CP 8 ), total deviation index, concordance correlation coefficient (CCC), measurement accuracy, and precision. For the liquid assay, there was satisfactory agreement among triplicate measurements made on different days at site 1, and high agreement based on accuracy and precision between duplicate measurements made on the same run at site 2. There was fair accuracy between measurements of the two sites performing the liquid assay, with CCCs below 0.6 for all phages tested. When compared to the plaque assay (performed once at site 3), there was less agreement between results of the liquid and plaque assays than between the two sites performing the liquid assay. Similar findings to the larger group were noted in the subset of 46 P . aeruginosa isolates from cystic fibrosis. Results of this study suggest that reproducibility of PST methods needs further development.

Abstract Background Increasing antimicrobial resistance combined with a lagging pipeline of novel antimicrobial compounds have resulted in a resurgence of interest in phage therapy. To select optimal phage or phage combinations for patients for whom phage therapy is considered, assessment of activity of a panel of phages against the patients’ bacterial isolate(s) should ideally be performed. Classical phage susceptibility testing methods (i.e., agar overlay) may be laborious, with expertise outside of normal training and competency of medical laboratory science staff needed. Content Adaptive Phage Therapeutics™ leveraged a commercially available phenotyping system (Biolog OmniLog®) to generate the PhageBank Susceptibility Test™, which uses a custom data analysis pipeline (PhageSelect™) to measure the delay in reaching log-phase metabolic activity (“hold time”) when a given isolate is challenged with a specific phage. The goal of this study was to preliminarily assess reproducibility of this approach by testing 2 bacterial species at 2 sites, APT and an academic site. Nineteen Escherichia coli phages were tested against 18 bacterial isolates, and 21 Staphylococcus aureus phages, against 11 bacterial isolates. Result comparisons were statistically excellent for E. coli (κ = 0.7990) and good/fair for S. aureus (κ = 0.6360). Summary The described method provides good/fair to excellent statistical reproducibility for assessment of phage susceptibility of 2 commonly encountered bacterial species.

Helicobacter pylori infection is mainly diagnosed noninvasively, with susceptibility testing traditionally requiring endoscopy. Treatment is empirical, with clarithromycin-based triple therapy recommended where resistance rates are below 15%. Rising rates of clarithromycin resistance, resulting in high therapy failure rates, are seen worldwide, but U.S. data are limited.

The taxonomic position of Yersinia kristensenii subsp. rochesterensis and Yersinia occitanica was re-evaluated by genomic analysis. Average nucleotide identity (ANI), digital DNA–DNA hybridization values, and phylogenetic analyses of the type strains indicate that Y. kristensenii subsp. rochesterensis and Y. occitanica are the same genospecies. Additionally, the overall genomic relatedness index (OGRI) values reveal that Y. kristensenii subsp. rochesterensis should be elevated to species status as Yersinia rochesterensis sp. nov.

AbstractBackgroundCurrent approaches in tracking Clostridioides difficile infection (CDI) and individualizing patient management are incompletely defined.MethodsWe recruited 468 subjects with CDI at Mayo Clinic Rochester between May and December 2016 and performed whole-genome sequencing (WGS) on C. difficile isolates from 397. WGS was also performed on isolates from a subset of the subjects at the time of a recurrence of infection. The sequence data were analyzed by determining core genome multilocus sequence type (cgMLST), with isolates grouped by allelic differences and the predicted ribotype.ResultsThere were no correlations between C. difficile isolates based either on cgMLST or ribotype groupings and CDI outcome. An epidemiologic assessment of hospitalized subjects harboring C. difficile isolates with ≤2 allelic differences, based on standard infection prevention and control assessment, revealed no evidence of person-to-person transmission. Interestingly, community-acquired CDI subjects in 40% of groups with ≤2 allelic differences resided within the same zip code. Among 18 subjects clinically classified as having recurrent CDI, WGS revealed 14 with initial and subsequent isolates differing by ≤2 allelic differences, suggesting a relapse of infection with the same initial strain, and 4 with isolates differing by >50 allelic differences, suggesting reinfection. Among the 5 subjects classified as having a reinfection based on the timing of recurrence, 3 had isolates with ≤2 allelic differences between them, suggesting a relapse, and 2 had isolates differing by >50 allelic differences, suggesting reinfection.ConclusionsOur findings point to potential transmission of C. difficile in the community. WGS better differentiates relapse from reinfection than do definitions based on the timing of recurrence.

Abstract Background Sonicate fluid (SF), a solution derived from vortexing and sonication of explanted cardiovascular implantable electronic devices (CIEDs), is a higher-yield specimen compared with swabs or tissues for culture-based detection of microorganisms associated with CIED infection. Despite this, SF culture fails to identify a causative organism in ~50% of cases. We aimed to evaluate the diagnostic performance of 16S ribosomal RNA gene (rRNA) polymerase chain reaction (PCR)/sequencing of SF and compare it with that of SF culture. Methods We identified 322 SF specimens from extracted CIEDs and reviewed clinical data for each patient. Subjects were classified as having or not having CIED infection. Cases were subcategorized as culture negative if no significant growth was reported from SF cultures and as culture positive if an organism was detected above predefined thresholds. 16S rRNA PCR/sequencing was performed, with the organisms identified reported according to Clinical and Laboratory Standards Institute guidelines for sequence data interpretation. Results A total of 278 SF samples corresponded to infected cases, of which 160 were culture positive and 118 culture negative. The remaining 44 were from noninfected cases, of which 2 were culture positive. Compared with SF culture, the sensitivity of 16S rRNA PCR/sequencing was higher (64% vs 57.5%, P = .003). 16S rRNA PCR/sequencing detected a potential pathogen in 28 of 118 culture-negative cases, identifying staphylococci in the majority (18/28). Conclusions 16S rRNA PCR/sequencing has higher sensitivity to detect bacteria in SF from extracted CIEDs than does SF culture.

Escherichia colibacteremia is caused mainly by sequence type complex 131 (STc131) and two clades within its fluoroquinolone-resistance-associatedH30 subclone,H30R1 andH30Rx. We examined clinical and molecular correlates ofE. colibacteremia in two geographically distinct centers.

Background Kingella kingae is a known cause of osteoarticular infections in children younger than 4 years of age, but it is not always recoverable in culture. Molecular methods are increasingly used for diagnosis. Methods To facilitate diagnosis of K. kingae septic arthritis, we developed a real-time polymerase chain reaction (PCR) assay for the detection of K. kingae that targets the repeat-in-toxin gene (rtxB). Results We present three pediatric patients with K. kingae septic arthritis at our institution who were diagnosed using the real-time PCR assay. All underwent arthrotomy with irrigation and debridement and were symptom-free after 3 weeks of therapy with β-lactam antibiotics. Cultures of synovial fluid or tissue grew K. kingae in two of three; K. kingae real-time PCR was positive in all three patients. In addition, 11 cases of K. kingae osteoarticular infection were diagnosed through Mayo Medical Laboratories using this assay. The limit of detection of the real-time PCR assay was 73.7 colony-forming unit (CFU)/µL for tissue and 1.3 CFU/µL for synovial fluid. Conclusions PCR-based detection methods are faster and more sensitive than conventional culture-based methods for the diagnosis of K. kingae osteoarticular infections in children.