We present a case of a 56-year-old Caucasian male who underwent a second kidney transplant in November 2023. Shortly thereafter, the patient developed acute pyelonephritis caused by Klebsiella oxytoca, treated initially with temocillin IV (2 g twice per day) and subsequently with amoxicillin-clavulanic acid orally (875 mg/125 mg three times per day) for a total of 14 days.

In January 2024, the patient presented to the emergency department with fever, which was attributed to recurrent pyelonephritis. Levofloxacin (500 mg once daily), started 3 weeks earlier in the context of an orthopedic infection, was continued. Two sets of blood cultures and a urine culture were taken at the emergency department, and the patient was admitted to the general ward. Therapy with IV temocillin was added for suspicion of pyelonephritis and a second set of blood cultures was collected at the time of fever.

The blood cultures were incubated in the automated BACTEC FX system (Becton Dickinson GmbH, Heidelberg, Germany). The two aerobic bottles of the last set flagged positive after 12 h. Gram-stain showed Gram-negative rods. Subcultures were made on a blood and MacConkey agar (BD) for overnight incubation and a short-term subculture was incubated on a blood agar for rapid identification after 5 h according to the method described by Verroken et al. [2]. Direct AST was done with the disk diffusion method according to EUCAST guidelines [3]. Additionally rapid AST was performed using the ASTar system, which performs fully automated microdilution AST directly from positive blood cultures with Gram-negative micro-organisms, in about 6 h [4].

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of the short-term subculture identified the Gram-negative rods in the blood culture as Klebsiella pneumoniae. The ASTar-system revealed a MIC of meropenem and ertapenem of 2 mg/L (Table 1), which is above the screening cut-off for CPE according to EUCAST [1]. The system does not test temocillin susceptibility, however. An OXA-48 K-SeT to detect OXA-48 was performed on the short-term subculture and was positive. Since OXA-48 type CPE is associated with high level resistance to temocillin [5], the therapy of the patient was urgently switched to ceftazidime-avibactam (1 g/0.25 g three times per day) and the patient was isolated. Figure 1 shows the timeline of antibiotic therapy and laboratory results. The next day, resistance to temocillin was confirmed by the disc diffusion method. We additionally performed the Xpert Carba-R assay on mature colonies, which was positive for OXA-48. Urine culture was also positive for OXA-48 type K. pneumoniae. The patient was treated 14 days with ceftazidime-avibactam with good clinical response.

Following this case, we performed a validation study to determine whether confirmatory CPE tests such as LFA or molecular assay can be reliably performed on short-term subcultures from positive blood cultures. Table 2 shows the validation protocol as well as the results of the validation study of OXA-48 K-SeT and Xpert Carba-R assay on short term cultures.