Patel, Robin

ABSTRACT The 16S rRNA sequences of enterococcal species E. faecium , E. faecalis , E. gallinarum , E. casseliflavus/flavescens , E. dispar , E. pseudoavium , E. sulfureus , E. malodoratus , E. raffinosus , E. cecorum , E. hirae , E. saccharolyticus , E. seriolicida , E. mundtii , E. avium , E. durans , E. columbae , and E. solitarius are presented herein. These data were utilized to confirm the species identification of two nonmotile E. gallinarum isolates which had been previously phenotypically identified as E. faecium . The implications of this finding are discussed.

Metagenomic shotgun sequencing for the identification of pathogens is being increasingly utilized as a diagnostic method. Interpretation of large and complicated data sets is a significant challenge, for which multiple commercial tools have been developed. Three commercial metagenomic shotgun sequencing tools, CosmosID, One Codex, and IDbyDNA, were compared to determine whether they result in similar interpretations of the same sequencing data.

Abstract Background Pharyngeal and rectal Neisseria gonorrhoeae and Chlamydia trachomatis play important roles in infection and antibacterial resistance transmission, but no US Food and Drug Administration (FDA)–cleared assays for detection at these sites existed prior to this study. The objective was to estimate performance of assays to detect those infections in pharyngeal and rectal specimens to support regulatory submission. Methods We performed a cross-sectional, single-visit study of adults seeking sexually transmitted infection testing at 9 clinics in 7 states. We collected pharyngeal and rectal swabs from participants. The primary outcome was positive and negative percent agreement for detection of N. gonorrhoeae and C. trachomatis for 3 investigational assays compared to a composite reference. Secondary outcomes included positivity as well as positive and negative predictive values and likelihood ratios. Subgroup analyses included outcomes by symptom status and sex. Results A total of 2598 participants (79% male) underwent testing. We observed N. gonorrhoeae positivity of 8.1% in the pharynx and 7.9% in the rectum and C. trachomatis positivity of 2.0% in the pharynx and 8.7% in the rectum. Positive percent agreement ranged from 84.8% to 96.5% for different anatomic site infection combinations, whereas negative percent agreement was 98.8% to 99.6%. Conclusions This study utilized a Master Protocol to generate diagnostic performance data for multiple assays from different manufacturers in a single study population, which ultimately supported first-in-class FDA clearance for extragenital assays. We observed very good positive percent agreement when compared to a composite reference method for the detection of both pharyngeal and rectal N. gonorrhoeae and C. trachomatis. Clinical Trials Registration NCT02870101.

The worldwide spread of multidrug-resistant Enterobacterales is a serious threat to public health. Here, we compared the MICs of plazomicin, amikacin, gentamicin, and tobramycin against 303 multinational multidrug-resistant Gram-negative bacilli. We followed Clinical and Laboratory Standards Institute (CLSI) guidelines and applied CLSI breakpoints as well as those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for amikacin, gentamicin, and tobramycin and of the U.S.

Abstract Background Sequence type 131 (EC-ST131) is a prevalent cause of extraintestinal Escherichia coli infection, including in neonates, and accounts for a majority of multidrug-resistant strains. Rare reports of neonatal unit outbreaks have emerged, with one linking the source to freshly expressed breast milk (BM) sharing. We report on premature twin girls whose infection was linked to their mother’s contaminated frozen BM. Methods Blood culture isolates were from twin girls born at 24–1/7 weeks’ gestation who developed severe sepsis caused by ampicillin- and gentamicin-resistant E. coli on days 11 (Baby A; died) and 8 (Baby B; survived) of life; both neonates had sterile blood cultures at birth, and received orogastric feeds using frozen BM provided by their mother. Five remaining frozen BM samples predating onset of sepsis were thawed and cultured; E. coli resistant to ampicillin and gentamicin was recovered from 1 collected on day 5 of life. DNA was extracted from cultured isolates using the Zymo Research Quick-DNA™ Fungal/Bacterial Miniprep kit, sequencing libraries prepared (Nextera® XT PE), and sequencing (Illumina MiSeq® with V2 2 X 250 bp chemistry) completed for the 2 blood and 1 BM isolates. Multilocus Sequence Typing (MLST) and core genome MLST (cgMLST) analyses were performed using SeqSphere+, version 5.1.0 (Ridom, Munster, DE) software, with 2513 alleles analyzed for cgMLST. Results The 2 blood and 1 BM isolates were typed as ST131 by MLST and were indistinguishable by cgMLST. Of the 2513 alleles queried, only 263 (10.5%) differed from the ST131 strain SCB34 (figure). Thus, our EC-ST131 cluster isolates belong to the same clonal complex as, but are genetically divergent from, SCB34. In addition to the E. coli described above isolated from a BM sample, all 5 frozen BM samples grew multiple morphotypes of coagulase-negative staphylococci. The mother had acute chorioamnionitis and was treated with intravenous ampicillin, gentamicin, and metronidazole for 48 hours immediately after delivery. Conclusion Despite frozen BM’s persistent antibacterial activity, it is a potential source of multidrug-resistant bacteria for neonates, as evidenced by this report of EC-ST131 neonatal sepsis in premature twins. Disclosures Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

Abstract Background Polymerase chain reaction (PCR)/sequencing targeting the 16S ribosomal RNA (rRNA) gene to detect bacteria in normally sterile tissues and fluids has become increasingly popular in clinical medicine. This culture-independent technique can detect bacteria that are nonviable or difficult to cultivate using conventional methods. The clinical value of this type of testing is not well defined. We aimed to assess the diagnostic value of 16S rRNA PCR/Sanger sequencing as a clinical diagnostic assay at Mayo Clinic. Methods This is an interim analysis of the first 173 of 478 patients who had 16S rRNA PCR/Sanger sequencing done on sterile tissues or fluids at our institution from April, 2017 to November, 2018 as part of routine clinical practice. Medical records are being retrospectively reviewed, with results compared with those of culture. Results We reviewed 207 specimens from 173 patients (musculoskeletal 79%, cardiovascular 7%, central nervous system 4%, other 9%) that underwent 16S rRNA PCR/Sanger sequencing by clinical request (Table 1). In 90% of these specimens, the test was pre-planned rather than added-on. Nine specimens were excluded from analysis, as cultures were not performed. Overall concordance of culture with PCR/sequencing was 81% (160/197; P < 0.0001). Of 44 culture-positive specimens, PCR detected the same bacterium in 21 (48%) (Table 2). 45% (20/44) of those with positive cultures and 46% of those with positive PCR/sequencing results had received prior antimicrobial therapy (Table 3). PCR was negative in 139/144 specimens that were culture-negative (97%). PCR/sequencing was helpful in detecting a putative bacterial pathogen in 4 patients with negative cultures (Table 4). Conclusion Overall, 16S rRNA PCR/Sanger sequencing improved diagnostic yield compared with culture in a minority of cases. The described assay is limited by its inability to detect polymicrobial infections, a technical limitation that could possibly be addressed using massive parallel sequencing. Careful selection of cases and a save and add-on approach may be more cost-effective than upfront testing, although this was requested in a minority of cases. Disclosures Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

Abstract Background We recently demonstrated that 16S rRNA PCR/sequencing performed on biofilms dislodged from extracted CIEDs into a salt solution—referred to as “sonicate fluid’ (SF)—may be used to detect pathogens in culture-negative CIED infection (Clinical Infectious Diseases, ciz266, doi:10.1093/cid/ciz266). The method we described included a DNA extraction/purification step, which can be time consuming and costly. Here, we evaluated an extraction-free approach to 16S rRNA gene PCR/sequencing. Methods 28 SF samples derived from explanted clinically-infected CIEDs were tested. Cases were categorized as “culture-positive” (C-P) if ≥20 cfu/10 mL were recovered, and as “culture-negative” (C-N), if <20 cfu/10 mL were detected in SF culture. The extraction-free method consisted of a single step of lysis at 1,000 rpm, 95°C for 5 minutes using an Eppendorf ThermoMixer®. DNA extraction (the comparator method) was performed using the ZymoBIOMICSTM Kit with modifications. Samples were processed using both methods, followed by amplification of the 16S rRNA gene and bidirectional Sanger sequencing. Crossing points (CPs) generated by the two approaches were compared. Organisms detected by the two PCR methods were compared with those detected with culture. Results Of the 28 samples tested, 13 were C-N and 15 C-P. The extraction-free method generated an amplicon in 13/15 C-P cases, with CPs ranging from 26 to 36 cycles vs. 100% (15/15) detected with the DNA extraction method and Cps of 19 to 32. Usable sequence length for the extraction-free method was of 359 (interquartile range, 307–390) vs. 390 (interquartile range, 308–396) base pairs with DNA extraction. Genus-level concordance between bacteria detected by culture in C-P samples and those found using the extraction-free and extraction methods was 92% (12/13) and 93% (14/15), respectively. Bacteria were detected by the extraction method in 2/13 C-N specimens, with none detected with an extraction-free method. Conclusion The described extraction-free method may be suitable for testing SF derived from CIEDs using 16S rRNA gene PCR/sequencing, saving time and cost. More studies are needed to establish clear cutoffs for interpretation of results and to assess for PCR inhibitors in the studied specimen-type. Disclosures Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

Abstract Background Orthopedic foreign body-associated infection can be difficult to treat due to the formation of biofilms protecting microorganisms from both antimicrobials and the immune system. Exebacase (EXE) is a phage-derived lysin which acts as a direct lytic agent by hydrolyzing the peptidoglycan cell wall of Staphylococcus aureus. In this study, the activity of EXE was evaluated in comparison to daptomycin against MRSA biofilms on orthopedic Kirschner wires (K-wires). Methods MRSA strain IDRL-6169 was studied; it has a MIC of 0.5 µg/mL for both daptomycin (DAP) and EXE. Biofilms were formed in 1 mL of 106 cfu/mL tryptic soy broth on 0.5x0.1 mm threaded stainless steel K-wires for 10 hours, after which the wires were removed from the media and placed into 0.04 mL of either DAP or EXE at 0 (vehicle only), 0.098, 0.98, or 9.8 mg/mL. DAP+EXE was also tested, each at 0.098 mg/mL. Bacteria were quantified after 0, 2, 4, 8, and 12 hours of incubation at 37ºC. Testing was performed in triplicate. Results were reported as log10 cfu/K-wire reduction relative to vehicle alone. A 3-log10 cfu/K-wire reduction was considered bactericidal. P-values were calculated using Kruskal–Wallis. Results The bacterial burden of vehicle alone ranged from 5.49- to 6.33-log10 cfu/K-wire at all time points. Bacterial reductions for each treatment compared with carrier solution are shown in the table. DAP showed no bactericidal activity. EXE showed bactericidal activity at all concentrations at all time points studied except 0.098 mg/mL at 8 hours. There was no significant difference between EXE at 0.098 and 0.98 mg/mL at any time point but EXE at 9.8 mg/mL did show superiority over the lower concentrations. DAP+EXE 0.098 mg/mL was bactericidal at all time points. Conclusion EXE showed a rapid effect against MRSA biofilms on orthopedic K-wires apparent within the first 2 hours of exposure and was more active than daptomycin alone at the same concentrations. Disclosures All authors: No reported disclosures.

Abstract Background Rapid blood culture diagnostics increase cost and have unclear benefit for patients with Gram-negative bacilli (GNB) bloodstream infections (BSIs). We conducted a multicenter, prospective randomized controlled trial (RAPIDS-GN), comparing outcomes of patients with GNB BSI who had blood culture testing with standard of care (SOC) culture and antibiotic susceptibility testing (AST) vs. rapid organism identification (ID) and phenotypic AST using the Accelerate Pheno System (AXDX). Methods Subjects with blood culture Gram stain showing GNB were randomized to receive SOC testing with antimicrobial stewardship review (AS) or AXDX plus SOC testing with AS, at two academic medical centers between October 2017 and October 2018. SOC testing included rapid MALDI-TOF mass spectrometry ID and agar dilution or broth microdilution AST. In a modified intention to treat analysis, subjects were excluded if: Gram stain was erroneous, culture was positive during off-hours, blood culture in the prior week had GNB, they were deceased/on comfort care, or admitted to a nonparticipating hospital. The primary outcome was time to first antibiotic modification within 72 hours after randomization. Subjects without antibiotic modifications were assigned a time of 72 hours. No censoring was observed. T-tests and Wilcoxon rank-sum tests were used for statistical analyses. Results Of 500 randomized subjects, 448 were included (226 SOC, 222 AXDX). Groups did not differ in baseline characteristics (Table 1). Median (IQR) hours to first antibiotic modification was faster in the AXDX vs. SOC group [8.6 (2.6, 27.6) vs. 14.9 (3.3, 41.1)], P = 0.02 (Figure 1). Median (IQR) hours to first Gram-negative antibiotic modification (including escalation and de-escalation) was faster in the AXDX than SOC group [17.4 (4.9, 72) vs. 42.1 (10.1, 72)], P < 0.001 (Figure 2). Groups did not differ in clinical outcomes (Table 2). Mean (S.D.) time to results was faster for AXDX than SOC for organism ID [2.7 (1.2) h vs. 15.6 (20.3) h, P < 0.001] and AST [13 (55.7) h vs. 54.6 (45.5) h, P < 0.001]. Conclusion In the largest trial to evaluate the clinical impact of a blood culture diagnostic for GNB BSI, we found that rapid organism ID and phenotypic AST led to faster changes in antibiotic therapy for Gram-negative bacteremia. Disclosures Ritu Banerjee, MD, PhD, Accelerate Diagnostics: Grant/Research Support; BioFire: Research Grant; Biomerieux: Research Grant; Roche: Research Grant Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.