Comparison of carbapenem-susceptible and carbapenem-resistant Enterobacterales at nine sites in the USA, 2013-2016: a resource for antimicrobial resistance investigators.

Carbapenem-resistant Enterobacterales (CRE) are an urgent public health threat. Genomic sequencing is an important tool for investigating CRE. Through the Division of Healthcare Quality Promotion Sentinel Surveillance system, we collected CRE and carbapenem-susceptible Enterobacterales (CSE) from nine clinical laboratories in the USA from 2013 to 2016 and analysed both phenotypic and genomic sequencing data for 680 isolates. We describe the molecular epidemiology and antimicrobial susceptibility testing (AST) data of this collection of isolates. We also performed a phenotype-genotype correlation for the carbapenems and evaluated the presence of virulence genes in Klebsiella pneumoniae complex isolates. These AST and genomic sequencing data can be used to compare and contrast CRE and CSE at these sites and serve as a resource for the antimicrobial resistance research community.

Reference Susceptibility Testing and Genomic Surveillance of Clostridioides difficile, United States, 2012-17.

BACKGROUND: Antimicrobial susceptibility testing (AST) is not routinely performed for Clostridioides difficile and data evaluating minimum inhibitory concentrations (MICs) are limited. We performed AST and whole genome sequencing (WGS) for 593 C. difficile isolates collected between 2012 and 2017 through the Centers for Disease Control and Prevention's Emerging Infections Program. METHODS: MICs to 6 antimicrobial agents (ceftriaxone, clindamycin, meropenem, metronidazole, moxifloxacin, and vancomycin) were determined using the reference agar dilution method according to Clinical and Laboratory Standards Institute guidelines. Whole genome sequencing was performed on all isolates to detect the presence of genes or mutations previously associated with resistance. RESULTS: Among all isolates, 98.5% displayed a vancomycin MIC ≤2 µg/mL and 97.3% displayed a metronidazole MIC ≤2 µg/mL. Ribotype 027 (RT027) isolates displayed higher vancomycin MICs (MIC50: 2 µg/mL; MIC90: 2 µg/mL) than non-RT027 isolates (MIC50: 0.5 µg/mL; MIC90: 1 µg/mL) (P < .01). No vanA/B genes were detected. RT027 isolates also showed higher MICs to clindamycin and moxifloxacin and were more likely to harbor associated resistance genes or mutations. CONCLUSIONS: Elevated MICs to antibiotics used for treatment of C. difficile infection were rare, and there was no increase in MICs over time. The lack of vanA/B genes or mutations consistently associated with elevated vancomycin MICs suggests there are multifactorial mechanisms of resistance. Ongoing surveillance of C. difficile using reference AST and WGS to monitor MIC trends and the presence of antibiotic resistance mechanisms is essential.

Risk-Factors for Exposure Associated With SARS-CoV-2 Detection After Recent Known or Potential COVID-19 Exposures Among Patients Seeking Medical Care at a Large Urban, Public Hospital in Fulton County, Georgia - A Cross-Sectional Investigation.

We aimed to describe frequency of COVID-19 exposure risk factors among patients presenting for medical care at an urban, public hospital serving mostly uninsured/Medicare/Medicaid clients and risk factors associated with SARS-CoV-2 infection. Consenting, adult patients seeking care at a public hospital from August to November 2020 were enrolled in this cross-sectional investigation. Saliva, anterior nasal and nasopharyngeal swabs were collected and tested for SARS-CoV-2 using RT-PCR. Participant demographics, close contact, and activities ≤14 days prior to enrollment were collected through interview. Logistic regression was used to identify risk factors associated with testing positive for SARS-CoV-2. Among 1,078 participants, 51.8% were male, 57.0% were aged ≥50 years, 81.3% were non-Hispanic Black, and 7.6% had positive SARS-CoV-2 tests. Only 2.7% reported COVID-19 close contact ≤14 days before enrollment; this group had 6.79 adjusted odds of testing positive (95%CI = 2.78-16.62) than those without a reported exposure. Among participants who did not report COVID-19 close contact, working in proximity to ≥10 people (adjusted OR = 2.17; 95%CI = 1.03-4.55), choir practice (adjusted OR = 11.85; 95%CI = 1.44-97.91), traveling on a plane (adjusted OR = 5.78; 95%CI = 1.70-19.68), and not participating in an essential indoor activity (i.e., grocery shopping, public transit use, or visiting a healthcare facility; adjusted OR = 2.15; 95%CI = 1.07-4.30) were associated with increased odds of testing positive. Among this population of mostly Black, non-Hispanic participants seeking care at a public hospital, we found several activities associated with testing positive for SARS-CoV-2 infection in addition to close contact with a case. Understanding high-risk activities for SARS-CoV-2 infection among different communities is important for issuing awareness and prevention strategies.

Specimen self-collection for SARS-CoV-2 testing: Patient performance and preferences-Atlanta, Georgia, August-October 2020.

Self-collected specimens can expand access to SARS-CoV-2 testing. At a large inner-city hospital 1,082 participants self-collected saliva and anterior nasal swab (ANS) samples before healthcare workers collected nasopharyngeal swab (NPS) samples on the same day. To characterize patient preferences for self-collection, this investigation explored ability, comfort, and ease of ANS and saliva self-collection for SARS-CoV-2 testing along with associated patient characteristics, including medical history and symptoms of COVID-19. With nearly all participants successfully submitting a specimen, favorable ratings from most participants (at least >79% in ease and comfort), and equivocal preference between saliva and ANS, self-collection is a viable SARS-CoV-2 testing option.

Effects of Patient Characteristics on Diagnostic Performance of Self-Collected Samples for SARS-CoV-2 Testing.

We evaluated the performance of self-collected anterior nasal swab (ANS) and saliva samples compared with healthcare worker-collected nasopharyngeal swab specimens used to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used the same PCR diagnostic panel to test all self-collected and healthcare worker-collected samples from participants at a public hospital in Atlanta, Georgia, USA. Among 1,076 participants, 51.9% were men, 57.1% were >50 years of age, 81.2% were Black (non-Hispanic), and 74.9% reported >1 chronic medical condition. In total, 8.0% tested positive for SARS-CoV-2. Compared with nasopharyngeal swab samples, ANS samples had a sensitivity of 59% and saliva samples a sensitivity of 68%. Among participants tested 3-7 days after symptom onset, ANS samples had a sensitivity of 80% and saliva samples a sensitivity of 85%. Sensitivity varied by specimen type and patient characteristics. These findings can help physicians interpret PCR results for SARS-CoV-2.

Characterization of Clostridioides difficile Isolates Available through the CDC & FDA Antibiotic Resistance Isolate Bank.

Thirty Clostridioides difficile isolates collected in 2016 through the Centers for Disease Control and Prevention Emerging Infections Program were selected for reference antimicrobial susceptibility testing and whole-genome sequencing. Here, we present the genetic characteristics of these isolates and announce their availability in the CDC & FDA Antibiotic Resistance Isolate Bank.

Sentinel Surveillance Reveals Emerging Daptomycin-Resistant ST736 Enterococcus faecium and Multiple Mechanisms of Linezolid Resistance in Enterococci in the United States.

Enterococcus faecalis and faecium with resistance to daptomycin and/or linezolid are emerging globally. We present the genomic characterization of daptomycin- and linezolid-resistant E. faecalis and E. faecium surveillance isolates from the United States, 2013-2016. Daptomycin resistance was low among E. faecalis (2/364, 0.5%) and E. faecium (17/344, 5%). The majority (71%, 12/17) of daptomycin-resistant E. faecium isolates belonged to the emerging ST736 clone and contained mutations in liaFSR and cls previously associated with resistance. However, 1/2 E. faecalis and 3/17 E. faecium did not contain these mutations previously associated with daptomycin resistance. Linezolid resistance was rare among E. faecalis (1/364, 0.3%) and E. faecium (2/344, 0.6%). These two E. faecium isolates, one of which was also resistant to daptomycin and vancomycin, contained the 23S rRNA nucleotide mutation (G2576T) associated with linezolid resistance. Long-read sequencing revealed the linezolid-resistant E. faecalis isolate contained chromosomal- and plasmid-encoded copies of optrA. The chromosomal optrA was located on the recently described Tn6674 multiresistance transposon. The second copy of optrA was encoded on an ∼65 kb mosaic plasmid, with component regions sharing high sequence identity to optrA-encoding multiresistance plasmids of animal origin. The optrA-encoding plasmid contained open reading frames predicted to encode proteins associated with a pheromone-responsive plasmid transfer system, and filter mating experiments confirmed the plasmid was conjugative. Continued surveillance of enterococci is necessary to assess the prevalence and trends of daptomycin and linezolid resistance in the United States, characterize resistance mechanisms and how they transfer, and monitor for emerging sequence types associated with resistance.

Rapid Nanopore Whole-Genome Sequencing for Anthrax Emergency Preparedness.

Human anthrax cases necessitate rapid response. We completed Bacillus anthracis nanopore whole-genome sequencing in our high-containment laboratory from a human anthrax isolate hours after receipt. The de novo assembled genome showed no evidence of known antimicrobial resistance genes or introduced plasmid(s). Same-day genomic characterization enhances public health emergency response.

Rapid Detection of Genetic Engineering, Structural Variation, and Antimicrobial Resistance Markers in Bacterial Biothreat Pathogens by Nanopore Sequencing.

Widespread release of Bacillus anthracis (anthrax) or Yersinia pestis (plague) would prompt a public health emergency. During an exposure event, high-quality whole genome sequencing (WGS) can identify genetic engineering, including the introduction of antimicrobial resistance (AMR) genes. Here, we developed rapid WGS laboratory and bioinformatics workflows using a long-read nanopore sequencer (MinION) for Y. pestis (6.5 h) and B. anthracis (8.5 h) and sequenced strains with different AMR profiles. Both salt-precipitation and silica-membrane extracted DNA were suitable for MinION WGS using both rapid and field library preparation methods. In replicate experiments, nanopore quality metrics were defined for genome assembly and mutation analysis. AMR markers were correctly detected and >99% coverage of chromosomes and plasmids was achieved using 100,000 raw sequencing reads. While chromosomes and large and small plasmids were accurately assembled, including novel multimeric forms of the Y. pestis virulence plasmid, pPCP1, MinION reads were error-prone, particularly in homopolymer regions. MinION sequencing holds promise as a practical, front-line strategy for on-site pathogen characterization to speed the public health response during a biothreat emergency.

Genome Sequences of Penicillin-Resistant Bacillus anthracis Strains.

Bacillus anthracis, the etiologic agent of anthrax, is characteristically susceptible to penicillin despite containing two chromosomal ß-lactamase genes. Few naturally occurring penicillin-resistant B. anthracis isolates have been reported. Here, we report the draft genome sequences for three penicillin-resistant B. anthracis strains, strain 32, UT308, and SK57.

Optical Screening for Rapid Antimicrobial Susceptibility Testing and for Observation of Phenotypic Diversity among Strains of the Genetically Clonal Species Bacillus anthracis.

During high-impact events involving Bacillus anthracis, such as the Amerithrax incident of 2001 or the anthrax outbreaks in Russia and Sweden in 2016, critical decisions to reduce morbidity and mortality include rapid selection and distribution of effective antimicrobial agents for treatment and postexposure prophylaxis. Detection of antimicrobial resistance currently relies on a conventional broth microdilution method that requires a 16- to 20-h incubation time for B. anthracis Advances in high-resolution optical screening offer a new technology to more rapidly evaluate antimicrobial susceptibility and to simultaneously assess the growth characteristics of an isolate. Herein, we describe a new method developed and evaluated as a rapid antimicrobial susceptibility test for B. anthracis This method is based on automated digital time-lapse microscopy to observe the growth and morphological effects of relevant antibiotics with an optical screening instrument, the oCelloScope. B. anthracis strains were monitored over time in the presence or absence of penicillin, ciprofloxacin, or doxycycline. Susceptibility to each antibiotic was determined in ≤4 h, 75 to 80% less than the time required for conventional methods. Time-lapse video imaging compiled from the optical screening images revealed unexpected differences in growth characteristics among strains of B. anthracis, which is considered to be a clonal organism. This technology provides a new approach for rapidly detecting phenotypic antimicrobial resistance and for documenting growth attributes that may be beneficial in the further characterization of individual strains.

Assuring the Quality of Next-Generation Sequencing in Clinical Microbiology and Public Health Laboratories.

Clinical microbiology and public health laboratories are beginning to utilize next-generation sequencing (NGS) for a range of applications. This technology has the potential to transform the field by providing approaches that will complement, or even replace, many conventional laboratory tests. While the benefits of NGS are significant, the complexities of these assays require an evolving set of standards to ensure testing quality. Regulatory and accreditation requirements, professional guidelines, and best practices that help ensure the quality of NGS-based tests are emerging. This review highlights currently available standards and guidelines for the implementation of NGS in the clinical and public health laboratory setting, and it includes considerations for NGS test validation, quality control procedures, proficiency testing, and reference materials.

Complete nucleotide sequences of plasmids pACK2 and pACK5 from Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus contains five plasmids, designated pACK1-pACK5. Recently, the nucleotide sequences of three of these plasmids, pACK1, pACK3, and pACK4, were reported. In order to complete the characterization of these five plasmids, the nucleotide sequences of the two remaining plasmids, pACK2 (37683 bp) and pACK5 (3191 bp), were determined. pACK5 is comprised of two regions, one with 85% identity at the nucleotide level to a region of pWBG1773 and another region with an ORF that shares no significant similarity to sequences previously described in GenBank. pACK2 encodes proteins for cadmium resistance and enhanced biofilm formation. The similarities at the nucleotide level among regions of the plasmids of S. simulans bv. staphylolyticus suggest that these plasmids have undergone multiple intermolecular rearrangements.

Complete nucleotide sequences of plasmids pACK1 and pACK3 from Staphylococcus simulans biovar staphylolyticus.

Staphylococcus simulans biovar staphylolyticus contains five plasmids designated pACK1-pACK5. The complete nucleotide sequences of pACK1 (55171bp) and pACK3 (28613bp) were determined and sequence comparison revealed that the entire pACK3 sequence is present on pACK1 (99.98% identical on the nucleotide level) with the sequence unique to pACK1 located between sin and blaZ, which are adjacent in pACK3. The common region contains the staphylococcal beta-lactamase transposon Tn552 and ORFs with similarity to genes encoding a serine-recombinase, enzymes involved in pantothenate biosynthesis, and components of the secA2 region involved in bacterial adherence to host tissues. The common region also contains a cluster of six ORFs that share no significant similarity to sequences previously described in GenBank. The region unique to pACK1, in addition to the genes for lysostaphin and lysostaphin resistance, contains ORFswith similarity to genes encoding a toxin-antitoxin addiction system and proteins involved in plasmid partitioning. The unique region also contains several ORFs that are similar to genes typically found on the chromosome such as those encoding catalase, ferrochelatase, and an enzyme involved in pantothenate biosynthesis. In pACK1, the ends of the unique region contain IS431 elements with direct repeats marking the points where the two plasmid sequences diverge. Several observations suggest that pACK1 was derived by insertion of the unique region into pACK3.