Amelia Bhatnagar

Metallo-beta-lactamase (MBL)-producing Enterobacterales are a global public health concern. Infections caused by these pathogens are often hard to treat, with few effective agents currently available. While aztreonam-avibactam only recently gained U.S. and European regulatory approval, laboratories have implemented laboratory-developed tests (LDTs) using existing aztreonam and ceftazidime-avibactam reagents. We sought to compare the performance of four phenotypic methods, up to three reagent manufacturers, and two media manufacturers. One hundred two MBL-producing Enterobacterales isolates were evaluated at two sites (Centers for Disease Control and Prevention [CDC] and Johns Hopkins University [JHU] School of Medicine). The performance of four methods was evaluated: broth disk elution (BDE), disk stacking (DS), gradient diffusion strip stacking (SS), and a strip-to-disk proximity (strip-disk) method, which were compared to reference broth microdilution (rBMD) tested in parallel. Respective category agreement for CDC and JHU was 92.2% and 96.1% for BDE, 51%–76.5% and 31%–49% for DS, 71.6%–86.3% and 83.3%–86.3% for SS, and 82.4%–87.3% and 80%–89.2% for the strip-disk method. The SS essential agreement ranged from 46.1% to 62.7% for JHU and 54.9% to 92.2% for CDC. Interlaboratory reproducibility was 93.1% for rBMD, 92.2% for BDE, 47.1%–53.1% for DS, 76.5%–80% for SS, and 87.3%–88.9% for strip-disk. BDE was the only method that exceeded acceptable performance of >90% accuracy and reproducibility. DS had the lowest and most variable performance between sites and among the different disk manufacturers used. LDTs for combination testing can be highly variable, and further work is needed to improve standardization and harmonization to achieve accurate and reliable results for clinical purposes. IMPORTANCE: Carbapenem-resistant Enterobacterales are classified as an urgent public health threat in the Centers for Disease Control and Prevention’s 2019 Antibiotic Resistance Threats Report. Among the most concerning resistance mechanisms are carbapenemase enzymes encoded on mobile genetic elements, which can spread rapidly between bacteria. Of the various carbapenemases, metallo-beta-lactamases are particularly challenging, as they confer resistance to nearly all beta-lactams, and current treatment options remain limited. While aztreonam-avibactam has been U.S. Food and Drug Administration (FDA)-approved for treating certain infections with limited or no alternative treatment options, no FDA-cleared susceptibility test is yet available. In this context, laboratory-developed methods, such as combination testing of aztreonam and ceftazidime-avibactam, play a critical role in guiding therapy. Our study evaluates several of the more common laboratory-developed tests compared to reference broth microdilution and found that broth disk elution was the most accurate; in contrast, other methods had variable performance, some demonstrating limited reliability and being prone to overcalling resistance.

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.

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.

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.

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.

Aztreonam/avibactam is a combination agent that shows promise in treating infections caused by highly antibiotic-resistant MBL-producing Enterobacterales. This combination can be achieved by combining two FDA-approved drugs: ceftazidime/avibactam and aztreonam. It is unknown whether ceftazidime in the combination ceftazidime/aztreonam/avibactam has a synergistic or antagonistic effect on the in vitro activity of aztreonam/avibactam by significantly increasing or decreasing the MIC. To determine whether increasing ceftazidime concentrations affect the MICs of aztreonam/avibactam alone. A custom 8 × 8 chequerboard broth microdilution (BMD) panel was made using a digital dispenser (Hewlett-Packard, Corvallis, OR, USA). The panel included orthogonal 2-fold dilution series of aztreonam and ceftazidime ranging from 0.5 to 64 mg/L. Avibactam concentration was kept constant at 4 mg/L throughout the chequerboard. Thirty-seven Enterobacterales isolates from the CDC & FDA Antibiotic Resistance Isolate Bank or CDC’s internal collection with intermediate or resistant interpretations to aztreonam and ceftazidime/avibactam were included for testing. All isolates harboured at least one of the following MBL genes: bla_IMP, bla_NDM or bla_VIM. Regardless of the concentration of ceftazidime, aztreonam/avibactam with ceftazidime MICs for all 37 isolates were within one 2-fold doubling dilution of the aztreonam/avibactam MIC. Ceftazidime, in the combination ceftazidime/avibactam/aztreonam, did not affect the in vitro activity of aztreonam/avibactam in this sample of isolates. These findings can help assure clinical and public health laboratories that testing of aztreonam/avibactam by BMD can act as a reliable surrogate test when the combination of ceftazidime/avibactam and aztreonam is being considered for treatment of highly antibiotic-resistant MBL-producing Enterobacterales.

On 6 February 2015, Kampala city authorities alerted the Ugandan Ministry of Health of a “strange disease” that killed one person and sickened dozens. We conducted an epidemiologic investigation to identify the nature of the disease, mode of transmission, and risk factors to inform timely and effective control measures. We defined a suspected case as onset of fever (≥37.5 °C) for more than 3 days with abdominal pain, headache, negative malaria test or failed anti-malaria treatment, and at least 2 of the following: diarrhea, nausea or vomiting, constipation, fatigue. A probable case was defined as a suspected case with a positive TUBEX® TF test. A confirmed case had blood culture yielding Salmonella Typhi. We conducted a case-control study to compare exposures of 33 suspected case-patients and 78 controls, and tested water and juice samples. From 17 February–12 June, we identified 10,230 suspected, 1038 probable, and 51 confirmed cases. Approximately 22.58% (7/31) of case-patients and 2.56% (2/78) of controls drank water sold in small plastic bags (ORM-H = 8.90; 95%CI = 1.60–49.00); 54.54% (18/33) of case-patients and 19.23% (15/78) of controls consumed locally-made drinks (ORM-H = 4.60; 95%CI: 1.90–11.00). All isolates were susceptible to ciprofloxacin and ceftriaxone. Water and juice samples exhibited evidence of fecal contamination. Contaminated water and street-vended beverages were likely vehicles of this outbreak. At our recommendation authorities closed unsafe water sources and supplied safe water to affected areas.