Allison C. Brown

Through their ability to hydrolyze carbapenems, Ambler class D beta-lactamases endanger patients by limiting the clinical efficacy of beta-lactam antimicrobials. Further, plasmid-mediated transmission can increase mobility of carbapenemase genes between bacteria and facilitate their spread between patients. In the United States and elsewhere, the plasmid-mediated Ambler class D carbapenemase genes bla _OXA-23-like , bla _{OXA-24/40-like} , and bla _OXA-58-like are commonly associated with Acinetobacter species and have rarely been reported outside of this genus. However, multiple recent international reports indicate detection of Enterobacterales isolates carrying Acinetobacter -associated class D carbapenemase genes. This evaluation aimed to provide insight into whether Enterobacterales harboring these class D genes may be circulating undetected in the United States, thereby signaling a need for adapting testing strategies to prioritize the detection of these potentially emerging public health threats. We analyzed whole-genome sequencing data generated through multiple Centers for Disease Control and Prevention (CDC) activities, including testing conducted across the Antimicrobial Resistance Laboratory Network, to determine whether any Enterobacterales isolates sequenced from 2017–2022 harbored Acinetobacter -associated class D carbapenemase genes. Among ∼10,000 predominantly carbapenem-resistant Enterobacterales isolates, we identified only a single Enterobacterales isolate harboring an Acinetobacter -associated class D gene – a Klebsiella pneumoniae isolate harboring a bla _OXA-23-like gene. Our findings suggest that bla _OXA-23-like , bla _{OXA-24/40-like} , and bla _OXA-58-like genes are rare among Enterobacterales isolates sequenced through CDC public health activities in the United States and do not warrant changes to current testing priorities at this time.

Carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are significant public health threats, particularly when harboring carbapenemases. Literature describing the frequencies and phenotypic and genotypic characteristics of isolates harboring multiple carbapenemase genes is limited. Using data collected from the Antimicrobial Resistance Laboratory Network (AR Lab Network) in 2018–2022, we describe CRE and CRPA isolates that harbor multiple acquired carbapenemase genes. Clinical laboratories submitted CRE and CRPA isolates to AR Lab Network public health laboratories for additional characterization that included antimicrobial susceptibility testing and detection of five targeted carbapenemase genes. Isolates were classified as non-carbapenemase producing (non-CP) when negative for carbapenemase production and all targeted carbapenemase genes, or positive for a single-CP (SCP) or multiple-carbapenemase (MCP) targeted gene. Among 79,799 CREs tested, 27,599 (35%) were SCP and 611 (1%) were MCP. MCP-CRE most often carried bla _KPC / bla _NDM ( n = 285, 47%). Both SCP-CRE and MCP-CRE were most commonly Klebsiella spp. Enterobacter spp. and Escherichia coli isolates harboring MCP were detected at slightly higher frequencies (18% and 15%; n = 109 and n = 88, respectively) than Enterobacter spp. and Escherichia coli isolates harboring SCP (13% and 13%; n = 3,653 and 3,471, respectively). The number of MCP-CRE detected increased from 54 of 5,105 (1%) in 2018 to 223 of 6,994 (3%) in 2022. Among 54,490 CRPA tested, 2% ( n = 1,249) were SCP and 31 were MCP. MCP-CRPA most often carried bla _VIM / bla _IMP ( n = 13, 42%). A higher proportion of MCP-CRE (97%, n = 330) isolates were categorized as resistant to meropenem, compared to SCP-CRE (79%; n = 11,227) and non-CP (13%; n = 2,683). Although MCP organisms represent a small proportion of total CP detected in the AR Lab Network, there is a need for continued monitoring and additional research. IMPORTANCE: Carbapenemase-producing organisms are of significant clinical and public health concerns, and rapid detection and containment of such threats are vital to preventing their spread. In this article, we used a collection of over 130,000 contemporary isolates to evaluate frequencies and phenotypic and genotypic properties of CRE and CRPA isolates harboring multiple carbapenemase genes across the United States, from 2018 to 2022. Of note, 95% and 100% of CRE and CRPA isolates co-harbored at least one metallo-β-lactamase gene, respectively, indicating a high proportion of isolates originating from patients with difficult-to-treat infections. Both clinical and public health professionals across the nation can use these data and key findings to better understand the molecular landscape of these isolates. Timely detection and control of these organisms are essential to combating the spread of antibiotic resistance and ensuring the availability of effective treatment options for patients.

Understanding the immune response kinetics to SARS-CoV-2 infection and COVID-19 vaccination is important in nursing home (NH) residents, a high-risk population. An observational longitudinal evaluation of 37 consenting vaccinated NH residents with/without SARS-CoV-2 infection from October 2020 to July 2022 was conducted to characterize the immune response to spike protein due to infection and/or mRNA COVID-19 vaccine. Antibodies (IgG) to SARS-CoV-2 full-length spike, nucleocapsid, and receptor binding domain protein antigens were measured, and surrogate virus neutralization capacity was assessed using Meso Scale Discovery immunoassays. The participant’s spike exposure status varied depending on the acquisition of infection or receipt of a vaccine dose. Longitudinal linear mixed effects modeling was used to describe trajectories based on the participant’s last infection or vaccination; the primary series mRNA COVID-19 vaccine was considered two spike exposures. Mean antibody titer values from participants who developed an infection post receipt of mRNA COVID-19 vaccine were compared with those who did not. In a subset of participants (n = 15), memory B cell (MBC) S-specific IgG (%S IgG) responses were assessed using an ELISPOT assay. The median age of the 37 participants at enrollment was 70.5 years; 30 (81%) had prior SARS-CoV-2 infection, and 76% received Pfizer-BioNTech and 24% Moderna homologous vaccines. After an observed augmented effect with each spike exposure, a decline in the immune response, including %S IgG MBCs, was observed over time; the percent decline decreased with increasing spike exposures. Participants who developed an infection at least two weeks post-receipt of a vaccine were observed to have lower humoral antibody levels than those who did not develop an infection post-receipt. These findings suggest that understanding the durability of immune responses in this vulnerable NH population can help inform public health policy regarding the timing of booster vaccinations as new variants display immune escape.

Acinetobacter baumannii is a Gram-negative bacillus that can cause severe and difficult-to-treat healthcare-associated infections. A. baumannii can harbor mobile genetic elements carrying genes that produce carbapenemase enzymes, further limiting therapeutic options for infections. In the United States, the Antimicrobial Resistance Laboratory Network (AR Lab Network) conducts sentinel surveillance of carbapenem-resistant Acinetobacter baumannii (CRAB). Participating clinical laboratories sent CRAB isolates to the AR Lab Network for characterization, including antimicrobial susceptibility testing and molecular detection of class A ( Klebsiella pneumoniae carbapenemase), class B (Active-on-Imipenem, New Delhi metallo-β-lactamase, and Verona integron-encoded metallo-β-lactamase), and class D (Oxacillinase, bla _OXA-23-like , bla _{OXA-24/40-like} , bla _OXA-48-like , and bla _OXA-58-like ) carbapenemase genes. During 2017‒2020, 6,026 CRAB isolates from 45 states were tested for targeted carbapenemase genes; 1% (64 of 5,481) of CRAB tested for targeted class A and class B genes were positive, but 83% (3,351 of 4,041) of CRAB tested for targeted class D genes were positive. The number of CRAB isolates carrying a class A or B gene increased from 2 of 312 (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.

In 2017, the Centers for Disease Control and Prevention (CDC) established the Antimicrobial Resistance Laboratory Network to improve domestic detection of multidrug-resistant organisms. CDC and four laboratories evaluated a commercial broth microdilution panel. Antimicrobial susceptibility testing using the Sensititre GN7F (ThermoFisher Scientific, Lenexa, KS) was evaluated by testing 100 CDC and Food and Drug Administration AR Isolate Bank isolates [40 Enterobacterales (ENT), 30 Pseudomonas aeruginosa (PSA), and 30 Acinetobacter baumannii (ACB)]. We assessed multiple amounts of transfer volume (TV) between the inoculum and tubed 11-mL cation-adjusted Mueller-Hinton broth: 1 µL [tribe Proteeae (P-tribe) only] and 10, 30, and 50 µL, resulting in respective CFU per milliter of 1 × 10 ⁴ , 1 × 10 ⁵ , 3 × 10 ⁵ , and 5 × 10 ⁵ . Four TV combinations were analyzed: standard (STD) [1 µL (P-tribe) and 10 µL], enhanced standard (E-STD) [1 µL (P-tribe) and 30 µL], 30 µL, and 50 µL. Essential agreement (EA), categorical agreement, major error (ME), and very major error (VME) were analyzed by organism then TVs. For ENT, the average EA across laboratories was 90%), and VMEs decreased. For PSA, EA improved as TVs increased; however, MEs also increased. For ACB, increased TVs provided slight EA improvements; all TVs yielded multiple VMEs and MEs. For ENT and ACB, Minimum inhibitory concentrations (MICs) trended downward using a 1 or 10 µL TV; there were no obvious MIC trends by TV for PSA. The public health and clinical consequences of missing resistance warrant increased TV of 30 µL for the GN7F, particularly for P-tribe, despite being considered “off-label” use.

There are limited data describing SARS-CoV-2–specific immune responses and their durability following infection and vaccination in nursing home residents. We conducted a prospective longitudinal evaluation of 11 consenting SARS-CoV-2–positive nursing home residents to evaluate the quantitative titers and durability of binding antibodies detected after SARS-CoV-2 infection and subsequent COVID-19 vaccination. The evaluation included nine visits over 150 days from October 25, 2020, through April 1, 2021. Visits included questionnaire administration, blood collection for serology, and paired anterior nasal specimen collection for testing by BinaxNOW^™ COVID-19 Ag Card (BinaxNOW), reverse transcription polymerase chain reaction (RT-PCR), and viral culture. We evaluated quantitative titers of binding SARS-CoV-2 antibodies post-infection and post-vaccination (beginning after the first dose of the primary series). The median age among participants was 74 years; one participant was immunocompromised. Of 10 participants with post-infection serology results, 9 (90%) had detectable Pan-Ig, IgG, and IgA antibodies, and 8 (80%) had detectable IgM antibodies. At first antibody detection post-infection, two-thirds (6/9, 67%) of participants were RT-PCR–positive, but none were culture- positive. Ten participants received vaccination; all had detectable Pan-Ig, IgG, and IgA antibodies through their final observation ≤90 days post-first dose. Post-vaccination geometric means of IgG titers were 10–200-fold higher than post-infection. Nursing home residents in this cohort mounted robust immune responses to SARS-CoV-2 post-infection and post-vaccination. The augmented antibody responses post-vaccination are potential indicators of enhanced protection that vaccination may confer on previously infected nursing home residents.

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: To characterize and compare severe acute respiratory coronavirus virus 2 (SARS-CoV-2)–specific immune responses in plasma and gingival crevicular fluid (GCF) from nursing home residents during and after natural infection.Design: Prospective cohort.Setting: Nursing home.Participants: SARS-CoV-2–infected nursing home residents.Methods: A convenience sample of 14 SARS-CoV-2–infected nursing home residents, enrolled 4–13 days after real-time reverse transcription polymerase chain reaction diagnosis, were followed for 42 days. After diagnosis, plasma SARS-CoV-2–specific pan-Immunoglobulin (Ig), IgG, IgA, IgM, and neutralizing antibodies were measured at 5 time points, and GCF SARS-CoV-2–specific IgG and IgA were measured at 4 time points.Results: All participants demonstrated immune responses to SARS-CoV-2 infection. Among 12 phlebotomized participants, plasma was positive for pan-Ig and IgG in all 12 participants. Neutralizing antibodies were positive in 11 participants; IgM was positive in 10 participants, and IgA was positive in 9 participants. Among 14 participants with GCF specimens, GCF was positive for IgG in 13 participants and for IgA in 12 participants. Immunoglobulin responses in plasma and GCF had similar kinetics; median times to peak antibody response were similar across specimen types (4 weeks for IgG; 3 weeks for IgA). Participants with pan-Ig, IgG, and IgA detected in plasma and GCF IgG remained positive throughout this evaluation, 46–55 days after diagnosis. All participants were viral-culture negative by the first detection of antibodies.Conclusions: Nursing home residents had detectable SARS-CoV-2 antibodies in plasma and GCF after infection. Kinetics of antibodies detected in GCF mirrored those from plasma. Noninvasive GCF may be useful for detecting and monitoring immunologic responses in populations unable or unwilling to be phlebotomized.

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.