Natália Kehl Moreira

Carbapenem-resistant Enterobacterales are a recognized health threat worldwide, due to high morbidity and mortality rates. After the COVID-19 pandemic, some geographic regions faced an increase in NDM-producing as well as NDM and KPC-co-producing Enterobacterales. Some of these species are intrinsically resistant to polymyxins, limiting significantly therapeutic options. This retrospective study analyzed 347 Enterobacterales resistant or intermediate to meropenem recovered between 2019 and 2023 from patients attending in a tertiary care hospital. The presence of carbapenemases and the susceptibility to ceftazidime-avibactam were assessed. Among members of Morganellaceae, 41.7% were resistant to meropenem while 58.2% were intermediate; most isolates (93.2%) produced NDM. On the other hand, among Serratia spp., resistance to meropenem was higher (78.7%) and isolates produced predominantly KPC (90.6%). Resistance to CAZ-AVI was not observed among isolates producing serine-carbapenemases. Our results reinforce the need for new therapeutic options to treat infections caused by gram-negative bacilli producing metallo-beta-lactamases, especially those intrinsically resistant to polymyxins.

Treating carbapenem-resistant Enterobacterales, mainly those producing metallo-β-lactamases (MBL) is a subject of major concern. The combination aztreonam-avibactam (AZA) was developed in an attempt to overcome this challenge, as avibactam inhibits KPC and other serine-β-lactamases, enabling aztreonam (ATM) to act against MBL. However, in scenarios where AZA is not licensed so far, the combination of ATM with ceftazidime-avibactam (CZA) is an alternative that has been proved in vivo efficacy and in vitro synergic activity. We propose the TurbiTest, an alternative method based on turbidity measurement of bacterial growth after a short incubation period (4 h) in the presence and absence of antibiotics to easily determine susceptibility to this combination. A total of 134 Enterobacterales harboring a variety of resistance genes (52 bla_KPC, 53 bla_NDM, 26 bla_KPC + bla_NDM, 2 bla_NDM + bla_{OXA−48−like}, 1 bla_{OXA−48−like}) as well as 11 Enterobacterales negative for carbapenemase genes by HRM-qPCR were evaluated for susceptibility to CZA, ATM, ATM-CZA and AZA. According to broth microdilution (BMD), 55.9% were resistant to CZA and 69.7% to ATM. All isolates were susceptible to ATM-CZA and AZA. Compared to BMD, TurbiTest from colonies (TTc) presented categorical agreement (CA) of 95.9% for CZA, 97.9% for ATM and ATM-CZA, and 99.3% for AZA. Fifty-eight spiked blood cultures were evaluated, with CA of 98.3% for CZA and ATM, and 100% for ATM-CZA and AZA. Besides, 41 clinical, consecutive Enterobacterales-positive blood cultures were tested (TTbc), with 100% of CA for CZA, 95.1% for ATM and 92.7% for ATM-CZA and AZA. TurbiTest is an inexpensive test that demonstrated a promising performance with considerably reduced turnaround time to results (less than 5 h). Interlaboratory reproducibility, as well as the performance with other bacterial population remain to be explored.

Context.—: Carbapenem-resistant Enterobacterales are disseminated worldwide and associated with infections with high rates of morbidity and mortality. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a useful tool for identification of pathogens directly from blood cultures in clinical microbiology laboratories. Furthermore, it has been applied for the detection of carbapenemase production, by evaluating carbapenem hydrolysis. Objective.—: To determine meropenem hydrolysis to detect carbapenemase production directly from positive blood cultures, using logRQ to establish a quantitative measure of hydrolysis. Design.—: We evaluated 100 Enterobacterales from positive blood cultures, with 81 carrying a carbapenemase gene (blaKPC, blaGES, blaNDM-1, blaIMP, blaVIM, and blaOXA-48-like), as determined by real-time multiplex polymerase chain reaction with high-resolution melting (HRM-qPCR). Bacterial proteins extracted from positive blood culture bottles were incubated in a meropenem solution (2–4 hours) followed by centrifugation for MALDI-TOF MS analysis. The intensity of peaks of the hydrolyzed and nonhydrolyzed forms were used to calculate the logRQ value. Results.—: Overall, sensitivity was 86.8% and specificity, 89.5%. Of note, sensitivity varied depending on enzyme type. For blaKPC-positive isolates, sensitivity was 97.9%, while it reduced significantly for blaNDM-1 and blaOXA-48-like isolates: 62.5% (10 of 16) and 66.7% (6 of 9), respectively. Indeed, logRQ was higher in blaKPC-positive isolates (0.37–1.97) than in blaNDM-1 (−1.37 to 0.83) and blaOXA-48-like isolates (−1.08 to 1.79). Conclusions.—: This is an inexpensive and rapid test to identify carbapenemase activity directly from blood culture bottles, which contributes to early adequate antimicrobial therapy and implementation of infection control measures.

Detecting carbapenemase-associated carbapenem resistance is a subject of major clinical and epidemiological concern as it influences therapeutic choice. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a means to assess bacterial resistance mechanisms. We aimed to detect the KPC enzyme directly from positive blood cultures using MALDI-TOF MS. To do so, 102 clinical Enterobacteria were evaluated, including 59 bla_KPC positives. Proteins were extracted using formic acid, isopropyl alcohol, and water (17:33:50) and spotted onto a steel target plate using the double-layer sinapinic acid technique. Two parameters were considered: (i) the visual detection of a clear peak with the expected KPC m/z and (ii) the evaluation of the relative intensity of the ions in the peak. A peak was observed in 56/59 bla_KPC-positive isolates (94.9% sensitivity), with no false-positive results (100% specificity). When considering intensity, with a cut-off ≥120 (a.u.), sensitivity was 94.9% and specificity was 95.3%. We proposed a “buffer” zone, with intermediate values of intensity (115 to 125) reaching 100% sensitivity and specificity. The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid, which may improve appropriate patient therapy and antimicrobial stewardship.

In this study, we evaluate a method for the KPC enzyme detection, using MALDI-TOF MS, for Enterobacterales. A total of 300 clinical Enterobacterales isolates were selected. The collection included 259 carbapenemase-producing (157 KPC and 102 non-KPC) and 41 carbapenemase non-producing isolates. Bacterial proteins were extracted from Mueller–Hinton agar plates using formic acid, isopropyl alcohol, and water (17:33:50). Samples were prepared with a double layer of synapinic acid. Analyses were performed using a Microflex LT mass spectrometer (Bruker Daltonics) and flexAnalysis 4.0 software (Bruker Daltonics). Statistical analyses were performed using SPSS Software. A distinctive peak at m/z 28,643–28,731 was found in all 157 KPC-producing isolates, and it was consistently absent in the 143 KPC non-producing group. KPC-producing peak intensities ranged from 77 to 3893. Considering an intensity cutoff value ≥ 120 for the presence of KPC, this methodology presented 98.09% and 97.90% of sensitivity and specificity, respectively.

Recently, new combinations of β-lactams and β-lactamase inhibitors became available, including ceftazidime-avibactam, and increased the ability to treat infections caused by carbapenem-resistant Enterobacterales (CRE). Despite the reduced time of clinical use, isolates expressing resistance to ceftazidime-avibactam have been reported, even during treatment or in patients with no previous contact with this drug. Here, we detailed review data on global ceftazidime-avibactam susceptibility, the mechanisms involved in resistance, and the molecular epidemiology of resistant isolates. Ceftazidime-avibactam susceptibility remains high (≥ 98.4%) among Enterobacterales worldwide, being lower among extended-spectrum β-lactamase (ESBL) producers and CRE. Alterations in class A β-lactamases are the major mechanism involved in ceftazidime-avibactam resistance, and mutations are mainly, but not exclusively, located in the Ω loop of these enzymes. Modifications in Klebsiella pneumoniae carbapenemase (KPC) 3 and KPC-2 have been observed by many authors, generating variants with different mutations, insertions, and/or deletions. Among these, the most commonly described is Asp179Tyr, both in KPC-3 (KPC-31 variant) and in KPC-2 (KPC-33 variant). Changes in membrane permeability and overexpression of efflux systems may also be associated with ceftazidime-avibactam resistance. Although several clones have been reported, ST258 with Asp179Tyr deserves special attention. Surveillance studies and rationale use are essential to retaining the activity of this and other antimicrobials against class A CRE.