A simple disk pre-diffusion test to predict in vitro aztreonam/avibactam activity against NDM-producing Klebsiella pneumoniae complex.

OBJECTIVES: The aim of this study was to describe a simple test to predict in vitro efficacy of aztreonam/avibactam (ATM-AVI) combination based on a pre-diffusion assay involving routinely available ceftazidime/avibactam (CAZ-AVI) and aztreonam (ATM) disks. METHODS: A total of 113 non-repetitive NDM-producing Klebsiella had the species identified by multiplex PCR. Minimum inhibitory concentrations (MICs) for ATM and ATM-AVI were determined by broth microdilution. For the combined disk pre-diffusion method, a disk containing ceftazidime 10 µg and avibactam 4 µg (CAZ-AVI) was applied on the surface of an uninoculated Mueller-Hinton agar plate. Following incubation for 2 h at 36°C, the disk was removed, the bacterial suspension was applied and a 30 µg ATM disk was placed precisely in the same position as the removed CAZ-AVI disk. Following incubation for 16-20 h, inhibition zone diameters were measured and correlated with ATM-AVI MICs. RESULTS: The distribution of species among the 113 isolates was 85 Klebsiella pneumoniae (75.2%), 19 Klebsiella quasipneumoniae (16.8%) and 9 Klebsiella variicola (8.0%). A total of 99 isolates had only blaNDM and 14 had both blaNDM and blaKPC genes. Regarding the isolates positive for blaNDM only, 38.4% were susceptible to ATM and 7.1% were susceptible, increased exposure. All isolates had ATM-AVI MICs of ≤1 mg/L, and the smallest inhibition zone diameter observed was 23 mm. CONCLUSION: Modified disk pre-diffusion can be used as a simple test to screen for ATM-AVI in vitro activity against Klebsiella, since ATM-AVI disks, gradient strips or microdilution panels are not commercially available.

Genotyping of paired KPC-producing Klebsiella pneumoniae isolates with and without divergent polymyxin B susceptibility profiles.

Polymyxins are still used mainly in treating infections caused by carbapenem-resistant Klebsiella pneumoniae worldwide. The most frequent mechanism of acquired resistance to polymyxins in Gram-negative bacilli is the occurrence of mutations in chromosomal genes regulating operons responsible for lipopolysaccharide modification. As we observed at Santa Casa de São Paulo hospital the occurrence of infections caused by isolates resistant to polymyxins in patients previously treated with this antimicrobial, and new infections caused by the same polymyxin-susceptible species, in this study, we aimed to determine the clonality of consecutive K. pneumoniae isolates from the same patients and characterize the molecular determinants of polymyxin resistance in paired or clonal isolates. A total of 24 pairs and one trio of K. pneumoniae isolates were included in this study. Species identification was achieved by mass spectrometry and multiplex PCR. Polymyxin B minimal inhibitory concentrations were determined by broth microdilution. Clonality was evaluated using pulsed-field gel electrophoresis. The presence of insertions in mgrB gene was tested by PCR, and mutations on pmrA, pmrB, phoP, and phoQ were evaluated by PCR and complete nucleotide sequencing. A fraction of 23.8% of strains resistant to polymyxin B had an insertion in mgrB. Amino acid substitution F204L in PmrB may be implicated in polymyxin resistance. Substitutions T246A and R256G in PmrB were not implicated in polymyxin resistance. In this study, polymyxin resistance after a first susceptible isolate was detected was most frequently due to an infection caused by a distinct clone.

Improved blood culture workflow for faster identification of KPC-producing Enterobacterales.

Carba-NP original report for blood cultures described the need of subculture and mechanical lysis before testing, reaching the turnaround time of approximately 4 hours for sample preparation. We tested 100 consecutive blood cultures positive for Gram-negative bacilli on the Gram stain from a large clinical laboratory. Bacterial pellets were prepared by centrifugation and submitted to Carba-NP and Blue-Carba tests and used further to prepare smears for Vitek MS. Results obtained with colonies grown on sheep blood agar using the same methodologies were used as the gold standard. Carbapenemase genes were confirmed by PCR and DNA sequencing. Vitek MS identified correctly 86% of the samples. Of note, 7% of the samples were incorrectly reported by the instrument as containing a single isolate. KPC-2 was the predominant carbapenemase detected. There was 100% concordance for both negative and positive results for Carba-NP. In contrast, for Blue-Carba the concordance for positive results was 92.8%, and 41% of strains negative for carbapenemases presented a yellowish color on control well turning the test non-interpretable. The turnaround time for sample preparation for preparing the pellet was 13 min, and no subculture or mechanical lysis is needed when detecting KPC production in Enterobacterales.