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.

Whole-Genome Sequencing Reveals Diversity of Carbapenem-Resistant Pseudomonas aeruginosa Collected through CDC's Emerging Infections Program, United States, 2016-2018.

The CDC's Emerging Infections Program (EIP) conducted population- and laboratory-based surveillance of US carbapenem-resistant Pseudomonas aeruginosa (CRPA) from 2016 through 2018. To characterize the pathotype, 1,019 isolates collected through this project underwent antimicrobial susceptibility testing and whole-genome sequencing. Sequenced genomes were classified using the seven-gene multilocus sequence typing (MLST) scheme and a core genome (cg)MLST scheme was used to determine phylogeny. Both chromosomal and horizontally transmitted mechanisms of carbapenem resistance were assessed. There were 336 sequence types (STs) among the 1,019 sequenced genomes, and the genomes varied by an average of 84.7% of the cgMLST alleles used. Mutations associated with dysfunction of the porin OprD were found in 888 (87.1%) of the genomes and were correlated with carbapenem resistance, and a machine learning model incorporating hundreds of genetic variations among the chromosomal mechanisms of resistance was able to classify resistant genomes. While only 7 (0.1%) isolates harbored carbapenemase genes, 66 (6.5%) had acquired non-carbapenemase ß-lactamase genes, and these were more likely to have OprD dysfunction and be resistant to all carbapenems tested. The genetic diversity demonstrates that the pathotype includes a variety of strains, and clones previously identified as high-risk make up only a minority of CRPA strains in the United States. The increased carbapenem resistance in isolates with acquired non-carbapenemase ß-lactamase genes suggests that horizontally transmitted mechanisms aside from carbapenemases themselves may be important drivers of the spread of carbapenem resistance in P. aeruginosa.

Comparison of the Risk of Recurrent Clostridioides Difficile Infections Among Patients in 2018 Versus 2013.

Among persons with an initial Clostridioides difficile infection (CDI) across 10 US sites in 2018 compared with 2013, 18.3% versus 21.1% had ≥1 recurrent CDI (rCDI) within 180 days. We observed a 16% lower adjusted risk of rCDI in 2018 versus 2013 (P < .0001).

Clinical and Genomic Epidemiology of mcr-9-Carrying Carbapenem-Resistant Enterobacterales Isolates in Metropolitan Atlanta, 2012 to 2017.

Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative infections. Recently, the ninth allele of the mobile colistin resistance (mcr) gene family, designated mcr-9, was reported. However, its clinical and public health significance remains unclear. We queried genomes of carbapenem-resistant Enterobacterales (CRE) for mcr-9 from a convenience sample of clinical isolates collected between 2012 and 2017 through the Georgia Emerging Infections Program, a population- and laboratory-based surveillance program. Isolates underwent phenotypic characterization and whole-genome sequencing. Phenotypic characteristics, genomic features, and clinical outcomes of mcr-9-positive and -negative CRE cases were then compared. Among 235 sequenced CRE genomes, 13 (6%) were found to harbor mcr-9, all of which were Enterobacter cloacae complex. The median MIC and rates of heteroresistance and inducible resistance to colistin were similar between mcr-9-positive and -negative isolates. However, rates of resistance were higher among mcr-9-positive isolates across most antibiotic classes. All cases had significant health care exposures. The 90-day mortality was similarly high in both mcr-9-positive (31%) and -negative (7%) CRE cases. Nucleotide identity and phylogenetic analysis did not reveal geotemporal clustering. mcr-9-positive isolates had a significantly higher number of median [range] antimicrobial resistance (AMR) genes (16 [4 to 22] versus 6 [2 to 15]; P < 0.001) than did mcr-9-negative isolates. Pangenome tests confirmed a significant association of mcr-9 detection with mobile genetic element and heavy metal resistance genes. Overall, the presence of mcr-9 was not associated with significant changes in colistin resistance or clinical outcomes, but continued genomic surveillance to monitor for emergence of AMR genes is warranted. IMPORTANCE Colistin is a last-resort antibiotic for multidrug-resistant Gram-negative infections. A recently described allele of the mobile colistin resistance (mcr) gene family, designated mcr-9, has been widely reported among Enterobacterales species. However, its clinical and public health significance remains unclear. We compared characteristics and outcomes of mcr-9-positive and -negative CRE cases. All cases were acquired in the health care setting and associated with a high rate of mortality. The presence of mcr-9 was not associated with significant changes in colistin resistance, heteroresistance, or inducible resistance but was associated with resistance to other antimicrobials and antimicrobial resistance (AMR), virulence, and heavy metal resistance (HMR) genes. Overall, the presence of mcr-9 was not associated with significant phenotypic changes or clinical outcomes. However, given the increase in AMR and HMR gene content and potential clinical impact, continued genomic surveillance of multidrug-resistant organisms to monitor for emergence of AMR genes is warranted.

Infectious Period of Severe Acute Respiratory Syndrome Coronavirus 2 in 17 Nursing Home Residents-Arkansas, June-August 2020.

BACKGROUND: To estimate the infectious period of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in older adults with underlying conditions, we assessed duration of coronavirus disease 2019 (COVID-19) symptoms, reverse-transcription polymerase chain reaction (RT-PCR) positivity, and culture positivity among nursing home residents. METHODS: We enrolled residents within 15 days of their first positive SARS-CoV-2 test (diagnosis) at an Arkansas facility from July 7 to 15, 2020 and instead them for 42 days. Every 3 days for 21 days and then weekly, we assessed COVID-19 symptoms, collected specimens (oropharyngeal, anterior nares, and saliva), and reviewed medical charts. Blood for serology was collected on days 0, 6, 12, 21, and 42. Infectivity was defined by positive culture. Duration of culture positivity was compared with duration of COVID-19 symptoms and RT-PCR positivity. Data were summarized using measures of central tendency, frequencies, and proportions. RESULTS: We enrolled 17 of 39 (44%) eligible residents. Median participant age was 82 years (range, 58-97 years). All had ≥3 underlying conditions. Median duration of RT-PCR positivity was 22 days (interquartile range [IQR], 8-31 days) from diagnosis; median duration of symptoms was 42 days (IQR, 28-49 days). Of 9 (53%) participants with any culture-positive specimens, 1 (11%) severely immunocompromised participant remained culture-positive 19 days from diagnosis; 8 of 9 (89%) were culture-positive ≤8 days from diagnosis. Seroconversion occurred in 12 of 12 (100%) surviving participants with ≥1 blood specimen; all participants were culture-negative before seroconversion. CONCLUSIONS: Duration of infectivity was considerably shorter than duration of symptoms and RT-PCR positivity. Severe immunocompromise may prolong SARS-CoV-2 infectivity. Seroconversion indicated noninfectivity in this cohort.

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.

A Pyrosequencing-Based Approach to High-Throughput Identification of Influenza A(H3N2) Virus Clades Harboring Antigenic Drift Variants.

The rapid evolution of influenza A(H3N2) viruses necessitates close monitoring of their antigenic properties so the emergence and spread of antigenic drift variants can be rapidly identified. Changes in hemagglutinin (HA) acquired by contemporary A(H3N2) viruses hinder antigenic characterization by traditional methods, thus complicating vaccine strain selection. Sequence-based approaches have been used to infer virus antigenicity; however, they are time consuming and mid-throughput. To facilitate virological surveillance and epidemiological studies, we developed and validated a pyrosequencing approach that enables identification of six HA clades of contemporary A(H3N2) viruses. The identification scheme of viruses of the H3 clades 3C.2, 3C.2a, 3C.2b, 3C.3, 3C.3a, and 3C.3b is based on the interrogation of five single nucleotide polymorphisms (SNPs) within three neighboring HA regions, namely 412 to 431, 465 to 481, and 559 to 571. Two bioinformatics tools, IdentiFire (Qiagen) and FireComb (developed in-house), were utilized to expedite pyrosequencing data analysis. The assay's analytical sensitivity was 10 focus forming units, and respiratory specimens with threshold cycle (CT) values of <34 typically produced good quality pyrograms. When applied to 120 A(H3N2) virus isolates and 27 respiratory specimens, the assay displayed 100% agreement with clades determined by HA sequencing coupled with phylogenetics. The multi-SNP analysis described here was readily adopted by another laboratory with pyrosequencing capabilities. The implementation of this approach enhanced the findings from virological surveillance and epidemiological studies between 2013 and 2016, which examined more than 3,000 A(H3N2) viruses.