Ten Clostridioides difficile isolates representing the top 10 ribotypes collected in 2016 through the Emerging Infections Program underwent long-read sequencing to obtain high-quality reference genome assemblies. These isolates are publicly available through the CDC & FDA Antibiotic Resistance Isolate Bank.

BACKGROUND: Understanding the epidemiology of carbapenem-resistant A. baumannii complex (CRAB) and the patients impacted is an important step towards informing better infection prevention and control practices and improving public health response. METHODS: Active, population-based surveillance was conducted for CRAB in 9 U.S. sites from January 1-December 31, 2019. Medical records were reviewed, isolates were collected and characterized including antimicrobial susceptibility testing and whole genome sequencing. RESULTS: Among 136 incident cases in 2019, 66 isolates were collected and characterized; 56.5% were from cases who were male, 54.5% were from persons of Black or African American race with non-Hispanic ethnicity, and the median age was 63.5 years. Most isolates, 77.2%, were isolated from urine, and 50.0% were collected in the outpatient setting; 72.7% of isolates harbored an acquired carbapenemase gene (aCP), predominantly bla(OXA-23) or bla(OXA-24/40); however, an isolate with bla(NDM) was identified. The antimicrobial agent with the most in vitro activity was cefiderocol (96.9% of isolates were susceptible). CONCLUSIONS: Our surveillance found that CRAB isolates in the U.S. commonly harbor an aCP, have an antimicrobial susceptibility profile that is defined as difficult-to-treat resistance, and epidemiologically are similar regardless of the presence of an aCP.

The Centers for Disease Control and Prevention has classified CRAB as an urgent public health threat. In this paper, we used a collection of >6,000 contemporary clinical isolates to evaluate the phenotypic and genotypic properties of CRAB detected in the United States. We describe the frequency of specific carbapenemase genes detected, antimicrobial susceptibility profiles, and the distribution of CRAB isolates categorized as multidrug resistant, extensively drug-resistant, or difficult to treat. We further discuss the proportion of isolates showing susceptibility to Food and Drug Administration-approved agents. Of note, 84% of CRAB tested harbored at least one class A, B, or D carbapenemase genes targeted for detection and 83% of these carbapenemase gene-positive CRAB were categorized as extensively drug resistant. Fifty-four percent of CRAB isolates without any of these carbapenemase genes detected were still extensively drug-resistant, indicating that infections caused by CRAB are highly resistant and pose a significant risk to patient safety regardless of the presence of one of these carbapenemase genes.

We thank Gonzales-Luna and colleagues [1] for their comments. We agree that laboratories must have access to accurate and standardized methods for antimicrobial susceptibility testing (AST) results to be clinically meaningful. The reference method for performing Clostridioides difficile AST is agar dilution according to Clinical and Laboratory Standards Institute (CLSI) guidelines [2]. The CLSI method for performing AST for anaerobic bacteria recommends that 5 μg/mL of hemin be incorporated into agar dilution plates and that the hemin stock solution should be protected from light and stored at 4°C–8°C for no longer than 1 month [2]. The susceptibility testing done by Gargis et al [3] was performed according to these guidelines, and the hemin stock solution was protected from light. | | Nevertheless, we read with interest the research in recent years [4–6] related to heme-dependent metronidazole resistance, including the reported association between isolates characterized as heme dependent and metronidazole resistant and the presence of a T to G mutation (PnimBG) in the −10 promoter region of the nitroimidazole reductase gene, nimB [5]. While Olaitan et al [5] found that not all heme-dependent metronidazole-resistant isolates contained the PnimBG mutation, Olaitan et al [5] indicate that most do; therefore, the presence of PnimBG may be predictive of resistance. We determined that the nimB mutation was present in 20% of our study isolates (116 of 593), of which 99% (115 of 116) belonged to RT027 (Table 1). The remaining isolate was RT014, the only RT014 isolate containing the PnimBG mutation among the 65 evaluated.

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.

Accurate antimicrobial susceptibility testing (AST) and reporting are essential for guiding appropriate therapy for patients and direction for public health prevention and control actions. A critical feature of AST reporting is the interpretation of AST results using clinical breakpoints for reporting as susceptible, susceptible-dose dependent, intermediate, or resistant. Breakpoints are subject to continuous adjustment and updating to best reflect current clinical data. These breakpoint changes can benefit patients and public health only if adopted in a timely manner. A recent survey identified that up to 70% of College of American Pathologists (CAP)-accredited U.S. laboratories and 45% of CAP-accredited laboratories outside the U.S. use various obsolete clinical breakpoints to interpret AST results to guide patient care. The reason for the ongoing use of obsolete breakpoints is multifactorial, including barriers encountered by laboratories, commercial AST device manufacturers, standards development organizations, and regulatory bodies alike. To begin to address this important patient safety issue, CAP implemented checklist requirements for CAP-accredited laboratories to ensure up-to-date clinical breakpoint use. Furthermore, the topic was discussed at the June 2022 American Society for Microbiology Clinical Microbiology Open (CMO) with various stakeholders to identify potential solutions. This minireview summarizes the breakpoint setting process in the U.S. and highlights solutions to close the gap between breakpoint revisions and implementation in clinical and public health laboratories. Solutions discussed include clarification of data requirements and minimum inhibitory concentration only reporting for regulatory clearance of AST devices, clinical data generation to close breakpoints gaps, advocacy, education, and greater dialogue between stakeholders.

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 2012-2017 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, thirteen (6%) were found to harbor mcr-9, all of which were Enterobacter cloacae complex. The median MIC, 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 healthcare 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 geo-temporal clustering. mcr-9 positive isolates had a significantly higher number of median [range] AMR genes (16 [4-22] vs. 6 [2-15]; p <0.001) compared to mcr-9 negative isolates. Pan genome 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.Competing Interest StatementThe authors have declared no competing interest.Funding StatementEIP Surveillance of the Multi-site Gram-Negative Surveillance Initiative (MuGSI) was funded through the Centers for Disease Control and Preventions Emerging Infections Program [U50CK000485].Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Georgia EIP surveillance activities are reviewed and approved by the Emory University Institutional Review Board.I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Oral fluids offer a non-invasive sampling method for the detection of antibodies. Quantification of IgA and IgG antibodies in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG antibodies against the prefusion-stabilized form of the SARS-CoV-2 spike protein. Normalization against total antibody isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 pre-pandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA: 95.5%; IgG: 89.7%) without compromising specificity (IgA: 99%; IgG: 97%). No cross reactivity with seasonal coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/mL and 0.30 ng/mL, respectively. Salivary IgA and IgG antibodies were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary antibody titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 weeks in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms has been adopted by many U.S. hospitals, but increasing chlorhexidine use has raised concerns about possible emergence of resistance. We sought to establish a broth microdilution method for determining chlorhexidine MICs and then used the method to evaluate chlorhexidine MICs for bacteria that can cause health care-associated infections. We adapted a broth microdilution method for determining chlorhexidine MICs, poured panels, established quality control ranges, and tested Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae complex isolates collected at three U.S. sites. Chlorhexidine MICs were determined for 535 isolates including 129 S. aureus, 156 E. coli, 142 K. pneumoniae, and 108 E. cloacae complex isolates. The respective MIC distributions for each species ranged from 1 to 8 mg/L (MIC(50) = 2 mg/L and MIC(90) = 4 mg/L), 1 to 64 mg/L (MIC(50) = 2 mg/L and MIC(90) = 4 mg/L), 4 to 64 mg/L (MIC(50) = 16 mg/L and MIC(90) = 32 mg/L), and 1 to >64 mg/L (MIC(50) = 16 mg/L and MIC(90) = 64 mg/L). We successfully adapted a broth microdilution procedure that several laboratories were able to use to determine the chlorhexidine MICs of bacterial isolates. This method could be used to investigate whether chlorhexidine MICs are increasing. IMPORTANCE Chlorhexidine bathing to prevent transmission of multidrug-resistant organisms and reduce health care-associated infections has been adopted by many hospitals. There is concern about the possible unintended consequences of using this agent widely. One possible unintended consequence is decreased susceptibility to chlorhexidine, but there are not readily available methods to perform this evaluation. We developed a method for chlorhexidine MIC testing that can be used to evaluate for possible unintended consequences.

Selective or cascade reporting (SR/CR) of antimicrobial susceptibility testing (AST) results is a strategy for antimicrobial stewardship. SR/CR is often achieved by suppressing AST results of secondary drugs in electronic laboratory reports. We assessed the extent of SR/CR and its impact on cumulative antibiograms (CAs) in a large cohort of U.S. hospitals submitting AST data to the CDC's National Healthcare Safety Network (NHSN) through electronic data exchange. The NHSN calls for hospitals to extract AST data from their electronic systems. We analyzed the AST reported for Escherichia coli (blood and urine) and Staphylococcus aureus (blood and lower respiratory tract [LRT]) isolates from April 2020 to March 2021, used AST reporting patterns to assign SR/CR reporting status for hospitals, and compared their CAs. Sensitivity analyses were done to account for those potentially extracted complete data. At least 35% and 41% of the hospitals had AST data that were suppressed in more than 20% blood isolates for E. coli and S. aureus isolates, respectively. At least 63% (blood) and 50% (urine) routinely reported ciprofloxacin or levofloxacin for E. coli isolates; and 60% (blood) and 59% (LRT) routinely reported vancomycin for S. aureus isolates. The distribution of CAs for many agents differed between high SR/CR and low- or non-SR/CR hospitals. Hospitals struggled to obtain complete AST data through electronic data exchange because of data suppression. Use of SR/CR can bias CAs if incomplete data are used. Technical solutions are needed for extracting complete AST results for public health surveillance. IMPORTANCE This study is the first to assess the extent of using selective and/or cascade antimicrobial susceptibility reporting for antimicrobial stewardship among U.S. hospitals and its impact on cumulative antibiograms in the context of electronic data exchange for national antimicrobial resistance surveillance.

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 2012-2017 through the Centers for Disease Control and Prevention's Emerging Infections Program. METHODS: MICs to six 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. WGS 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 < 0.01). 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.

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 β-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 β-lactamase genes suggests that horizontally transmitted mechanisms aside from carbapenemases themselves may be important drivers of the spread of carbapenem resistance in P. aeruginosa.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are usually healthcare-associated but are also emerging in the community. METHODS: Active, population-based surveillance was conducted to identify case-patients with cultures positive for Enterobacterales not susceptible to a carbapenem (excluding ertapenem) and resistant to all third-generation cephalosporins tested at 8 US sites from January 2012 to December 2015. Medical records were used to classify cases as health care-associated, or as community-associated (CA) if a patient had no known health care risk factors and a culture was collected <3 days after hospital admission. Enterobacterales isolates from selected cases were submitted to CDC for whole genome sequencing. RESULTS: We identified 1499 CRE cases in 1194 case-patients; 149 cases (10%) in 139 case-patients were CA. The incidence of CRE cases per 100,000 population was 2.96 (95% CI: 2.81, 3.11) overall and 0.29 (95% CI: 0.25, 0.35) for CA-CRE. Most CA-CRE cases were in White persons (73%), females (84%) and identified from urine cultures (98%). Among the 12 sequenced CA-CRE isolates, 5 (42%) harbored a carbapenemase gene. CONCLUSIONS: Ten percent of CRE cases were CA; some isolates from CA-CRE cases harbored carbapenemase genes. Continued CRE surveillance in the community is critical to monitor emergence outside of traditional health care settings.

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.

TO THE EDITORWe read with concern the report by Darkoh and colleagues describing vancomycin resistance in Clostridioides difficile isolates recovered from patients in Houston, Texas, and Nairobi, Kenya [1]. The authors suggested that vancomycin resistance, which they found to be common, may lead to therapeutic failure for patients infected with C. difficile and that routine antimicrobial susceptibility testing (AST) should be expanded. The authors report that 26% of C. difficile toxin gene-positive patients in Houston and 67% of sequential hospitalized patients in Nairobi with acute diarrhea had growth on primary screening media containing 4g/mL vancomycin. This alarmingly high proportion of a vancomycin resistant phenotype exceeds initial treatment failure rates reported in the literature [2], calling into question the reliability of these findings.

Healthcare-associated carbapenem-resistant Acinetobacter baumannii (CRAB) infections are a serious threat associated with global epidemic clones and a variety of carbapenemase gene classes. In this study, we describe the molecular epidemiology, including whole-genome sequencing analysis and antimicrobial susceptibility profiles of 92 selected, nonredundant CRAB collected through public health efforts in the United States from 2013 to 2017. Among the 92 isolates, the Oxford (OX) multilocus sequence typing scheme identified 30 sequence types (STs); the majority of isolates (n = 59, 64%) represented STs belonging to the international clonal complex 92 (CC92(OX)). Among these, ST208(OX) (n = 21) and ST281(OX) (n = 20) were the most common. All isolates carried an OXA-type carbapenemase gene, comprising 20 alleles. Ninety isolates (98%) encoded an intrinsic OXA-51-like enzyme; 67 (73%) harbored an additional acquired bla(OXA) gene, most commonly bla(OXA-23) (n = 45; 49%). Compared with isolates harboring only intrinsic oxacillinase genes, acquired bla(OXA) gene presence was associated with higher prevalence of resistance and a higher median minimum inhibitory concentration to the carbapenem imipenem (64 μg/mL vs. 8 μg/mL), and antibiotics from other drug classes, including penicillin, aminoglycosides, cephalosporins, and polymyxins. These data illustrate the wide distribution of CC92(OX) and high prevalence of acquired bla(OXA) carbapenemase genes among CRAB 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.

Oral fluids offer a noninvasive sampling method for the detection of Abs. Quantification of IgA and IgG Abs in saliva allows studies of the mucosal and systemic immune response after natural infection or vaccination. We developed and validated an enzyme immunoassay (EIA) to detect and quantify salivary IgA and IgG Abs against the prefusion-stabilized form of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein expressed in suspension-adapted HEK-293 cells. Normalization against total Ab isotype was performed to account for specimen differences, such as collection time and sample volume. Saliva samples collected from 187 SARS-CoV-2 confirmed cases enrolled in 2 cohorts and 373 prepandemic saliva samples were tested. The sensitivity of both EIAs was high (IgA, 95.5%; IgG, 89.7%) without compromising specificity (IgA, 99%; IgG, 97%). No cross-reactivity with endemic coronaviruses was observed. The limit of detection for SARS-CoV-2 salivary IgA and IgG assays were 1.98 ng/ml and 0.30 ng/ml, respectively. Salivary IgA and IgG Abs were detected earlier in patients with mild COVID-19 symptoms than in severe cases. However, severe cases showed higher salivary Ab titers than those with a mild infection. Salivary IgA titers quickly decreased after 6 wk in mild cases but remained detectable until at least week 10 in severe cases. Salivary IgG titers remained high for all patients, regardless of disease severity. In conclusion, EIAs for both IgA and IgG had high specificity and sensitivity for the confirmation of current or recent SARS-CoV-2 infections and evaluation of the IgA and IgG immune response.

OBJECTIVE: The incidence of infections from extended-spectrum β-lactamase (ESBL)-producing Enterobacterales (ESBL-E) is increasing in the United States. We describe the epidemiology of ESBL-E at 5 Emerging Infections Program (EIP) sites. METHODS: During October-December 2017, we piloted active laboratory- and population-based (New York, New Mexico, Tennessee) or sentinel (Colorado, Georgia) ESBL-E surveillance. An incident case was the first isolation from normally sterile body sites or urine of Escherichia coli or Klebsiella pneumoniae/oxytoca resistant to ≥1 extended-spectrum cephalosporin and nonresistant to all carbapenems tested at a clinical laboratory from a surveillance area resident in a 30-day period. Demographic and clinical data were obtained from medical records. The Centers for Disease Control and Prevention (CDC) performed reference antimicrobial susceptibility testing and whole-genome sequencing on a convenience sample of case isolates. RESULTS: We identified 884 incident cases. The estimated annual incidence in sites conducting population-based surveillance was 199.7 per 100,000 population. Overall, 800 isolates (96%) were from urine, and 790 (89%) were E. coli. Also, 393 cases (47%) were community-associated. Among 136 isolates (15%) tested at the CDC, 122 (90%) met the surveillance definition phenotype; 114 (93%) of 122 were shown to be ESBL producers by clavulanate testing. In total, 111 (97%) of confirmed ESBL producers harbored a blaCTX-M gene. Among ESBL-producing E. coli isolates, 52 (54%) were ST131; 44% of these cases were community associated. CONCLUSIONS: The burden of ESBL-E was high across surveillance sites, with nearly half of cases acquired in the community. EIP has implemented ongoing ESBL-E surveillance to inform prevention efforts, particularly in the community and to watch for the emergence of new ESBL-E strains.

Carbapenem-resistant Enterobacterales (CRE) are a growing public health concern due to resistance to multiple antibiotics and potential to cause health care-associated infections with high mortality. Carbapenemase-producing CRE are of particular concern given that carbapenemase-encoding genes often are located on mobile genetic elements that may spread between different organisms and species. In this study, we performed phenotypic and genotypic characterization of CRE collected at eight U.S. sites participating in active population- and laboratory-based surveillance of carbapenem-resistant organisms. Among 421 CRE tested, the majority were isolated from urine (n = 349, 83%). Klebsiella pneumoniae was the most common organism (n = 265, 63%), followed by Enterobacter cloacae complex (n = 77, 18%) and Escherichia coli (n = 50, 12%). Of 419 isolates analyzed by whole genome sequencing, 307 (73%) harbored a carbapenemase gene; variants of bla(KPC) predominated (n = 299, 97%). The occurrence of carbapenemase-producing K. pneumoniae, E. cloacae complex, and E. coli varied by region; the predominant sequence type within each genus was ST258, ST171, and ST131, respectively. None of the carbapenemase-producing CRE isolates displayed resistance to all antimicrobials tested; susceptibility to amikacin and tigecycline was generally retained.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are highly antibiotic-resistant bacteria. Whether CRE resistant only to ertapenem among carbapenems (ertapenem "mono-resistant") represent a unique CRE subset with regards to risk factors, carbapenemase genes, and outcomes is unknown. METHODS: We analyzed surveillance data from 9 CDC Emerging Infections Program (EIP) sites. A case was the first isolation of a carbapenem-resistant Enterobacter cloacae complex, Escherichia coli, Klebsiella aerogenes, K. oxytoca, K. pneumoniae, or K. variicola from a normally sterile site or urine in an EIP catchment area resident in 2016-2017. We compared risk factors, carbapenemase genes, antibiotic susceptibility, and mortality of ertapenem "mono-resistant" cases to "other" CRE cases (resistant to1 carbapenem other than ertapenem) and analyzed risk factors for mortality. RESULTS: Of 2009 cases, 1249 (62.2%) were ertapenem-mono-resistant and 760 (37.8%) were other CRE. Ertapenem-mono-resistant CRE cases were more frequently80 years old (29.1% vs 19.5%; P<.0001) and female (67.9% vs 59.0%; P<.0001). Ertapenem-mono-resistant isolates were more likely to be Enterobacter cloacae complex (48.4% vs 15.4%; P<.0001) but less likely to be isolated from a normally sterile site (7.1% vs 11.7%; P<.01) or to have a carbapenemase gene (2.4% vs 47.4%; P<.0001). Ertapenem-mono-resistance was not associated with 90-day mortality in logistic regression models. Carbapenemase-positive isolates were associated with mortality (odds ratio, 1.93; 95% CI, 1.30-2.86). CONCLUSIONS: Ertapenem-mono-resistant CRE rarely have carbapenemase genes and have distinct clinical and microbiologic characteristics from other CRE. These findings may inform antibiotic choice and infection prevention practices, particularly when carbapenemase testing is not available.

Carbapenemase gene-positive (CP) Gram-negative bacilli are of significant clinical and public health concern. Their rapid detection and containment are critical to preventing their spread and additional infections they can cause. To this end, CDC developed the Antibiotic Resistance Laboratory Network (AR Lab Network), in which public health laboratories across all 50 states, several cities, and Puerto Rico characterize clinical isolates of carbapenem-resistant Enterobacterales (CRE), Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB), and conduct colonization screens to detect the presence of mobile carbapenemase genes. In its first three years, the AR Lab Network tested 76,887 isolates and 31,001 rectal swab colonization screens. Targeted carbapenemase genes (bla(KPC), bla(NDM), bla(OXA-48-like), bla(VIM), or bla(IMP)) were detected by PCR in 35% of CRE, 2% of CRPA, <1% of CRAB, and 8% of colonization screens tested, respectively. bla(KPC) and bla(VIM) were the most common CP-CRE and CP-CRPA, respectively, but regional differences in the frequency of carbapenemase genes detected were apparent. In CRE and CRPA isolates tested for carbapenemase production and the presence of the targeted genes, 97% had concordant results; 3% of CRE and 2% of CRPA were carbapenemase production-positive but PCR-negative for those genes. Isolates harboring bla(NDM) showed the highest frequency of resistance across the carbapenems tested and those harboring bla(IMP) and bla(OXA-48-like) genes showed the lowest frequency of carbapenem resistance. The AR Lab Network provides a national snapshot of rare and emerging carbapenemase genes, delivering data to inform public health actions to limit the spread of these antibiotic resistance threats.

Diagnostic stewardship means ordering the right tests, for the right patient at the right time to inform optimal clinical care. Diagnostic stewardship is an integral part of antibiotic stewardship efforts to optimize antibiotic use and improve patient outcomes, including reductions in antibiotic resistance, and treatment of sepsis. CDC's Division of Healthcare Quality Promotion (DHQP) hosted a meeting on improving patient safety through diagnostic stewardship with a focus on the use of the laboratory. The meeting identified emerging issues in the field of diagnostic stewardship, raised awareness of these issues among stakeholders, and discussed strategies and interventions to address the issues-all with an emphasis on improved outcomes and patient safety. This white paper summarizes the key takeaways of the meeting including needs for diagnostic stewardship implementation, promising future avenues for diagnostic stewardship implementation, and areas of needed research.

BACKGROUND: Antimicrobial resistance is a global health emergency. Persons colonized with multidrug-resistant organisms (MDROs) are at risk for developing subsequent multidrug-resistant infections, as colonization represents an important precursor to invasive infection. Despite reports documenting the worldwide dissemination of MDROs, fundamental questions remain regarding the burden of resistance, metrics to measure prevalence, and determinants of spread. We describe a multi-site colonization survey protocol that aims to quantify the population-based prevalence and associated risk factors for colonization with high-threat MDROs among community dwelling participants and patients admitted to hospitals within a defined population-catchment area. METHODS: Researchers in five countries (Bangladesh, Chile, Guatemala, Kenya, and India) will conduct a cross-sectional, population-based prevalence survey consisting of a risk factor questionnaire and collection of specimens to evaluate colonization with three high-threat MDROs: extended-spectrum cephalosporin-resistant Enterobacteriaceae (ESCrE), carbapenem-resistant Enterobacteriaceae (CRE), and methicillin-resistant Staphylococcus aureus (MRSA). Healthy adults residing in a household within the sampling area will be enrolled in addition to eligible hospitalized adults. Colonizing isolates of these MDROs will be compared by multilocus sequence typing (MLST) to routinely collected invasive clinical isolates, where available, to determine potential pathogenicity. A colonizing MDRO isolate will be categorized as potentially pathogenic if the MLST pattern of the colonizing isolate matches the MLST pattern of an invasive clinical isolate. The outcomes of this study will be estimates of the population-based prevalence of colonization with ESCrE, CRE, and MRSA; determination of the proportion of colonizing ESCrE, CRE, and MRSA with pathogenic characteristics based on MLST; identification of factors independently associated with ESCrE, CRE, and MRSA colonization; and creation an archive of ESCrE, CRE, and MRSA isolates for future study. DISCUSSION: This is the first study to use a common protocol to evaluate population-based prevalence and risk factors associated with MDRO colonization among community-dwelling and hospitalized adults in multiple countries with diverse epidemiological conditions, including low- and middle-income settings. The results will be used to better describe the global epidemiology of MDROs and guide the development of mitigation strategies in both community and healthcare settings. These standardized baseline surveys can also inform future studies seeking to further characterize MDRO epidemiology globally.

Replication-competent virus has not been detected in individuals with mild to moderate COVID-19 more than 10 days after symptom onset. It is unknown whether these findings apply to nursing home residents. Of 273 specimens collected from nursing home residents >10 days from the initial positive test, none were culture positive.

Aztreonam-avibactam is a drug combination pending phase 3 clinical trials and is suggested for treatment of severe infections caused by metallo-beta-lactamase (MBL)-producing Enterobacterales by combining ceftazidime-avibactam and aztreonam. Beginning in 2019, four Antibiotic Resistance Laboratory Network regional laboratories offered aztreonam-avibactam susceptibility testing by broth microdilution. For 64 clinical isolates tested, the MIC(50) and MIC(90) of aztreonam-avibactam were 0.5/4 μg/mL and 8/4 μg/mL, respectively. Aztreonam-avibactam displayed potent in vitro activity against the MBL-producing Enterobacterales tested.

Infections caused by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are a significant cause of morbidity and healthcare costs. Globally, the prevailing clonal type is ST131 in association with the blaCTX-M-15 β-lactamase gene. However, other ESBLs such as blaCTX-M-14 and blaCTX-M-27 can also be prevalent in some regions. We identified ST38 ESBL-producing E. coli from different regions in the US which carry blaCTX-M-27 embedded on two distinct plasmid types, suggesting the potential emergence of new ESBL lineages.

Background: Detection of carbapenem-resistant Pseudomonas aeruginosa (CRPA) with carbapenamase-producing (CP) genes is critical for preventing transmission. Our objective was to assess whether certain antimicrobial susceptibility testing (AST) profiles can efficiently identify CP-CRPA.Methods: We defined CRPA as P. aeruginosa with imipenem or meropenem MICs of ≥8μg/ml; CP-CRPA were CRPA with CP genes (bla (KPC)/bla (IMP)/bla (NDM)/bla (VIM)). We assessed the sensitivity and specificity of AST profiles to detect CP-CRPA among CRPA collected by CDC's Antibiotic Resistance Laboratory Network (AR Lab Network) and the Emerging Infections Program (EIP) during 2017-2019.Results: Three percent (195/6192) of AR Lab Network CRPA were CP-CRPA. Among CRPA, adding not susceptible (NS) to cefepime or ceftazidime to the definition had 91% sensitivity and 50% specificity for identifying CP-CRPA; NS to ceftolozane-tazobactam had 100% sensitivity and 86% specificity. Of 965 EIP CRPA evaluated for CP genes, seven CP-CRPA were identified; 6 of 7 were NS to cefepime and ceftazidime, and all 7 were NS to ceftolozane-tazobactam. Among 4182 EIP isolates, clinical laboratory AST results were available for 96% for cefepime, 80% for ceftazidime, and 4% for ceftolozane-tazobactam. The number of CRPA needed to test (NNT) to identify one CP-CRPA decreased from 138 to 64 if the definition of NS to cefepime or ceftazidime was used and to 7 with NS to ceftolozane-tazobactam.Conclusion: Adding not susceptible to cefepime or ceftazidime to CRPA carbapenemase testing criteria would reduce the NNT by half and can be implemented in most clinical laboratories; adding not susceptible to ceftolozane-tazobactam could be even more predictive once AST for this drug is more widely available.

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.