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Objectives: To evaluate the in vitro activity of ceftazidime/avibactam, meropenem/vaborbactam, imipenem/relebactam and comparators against KPC-producing Klebsiella pneumoniae (KPC-Kp) clinical isolates collected from a multicentre study in Italy (2022-23) and genomic characterization of the molecular mechanisms causing resistance., Methods: Consecutive KPC-Kp isolates from blood cultures (n = 264) were collected from 14 hospital centres in the period 2022-23. Antimicrobial susceptibility testing was performed using broth microdilution. WGS was used to investigate KPC-Kp strains resistant to the new approved β-lactam/β-lactam inhibitor combinations (BLICs)., Results: Overall, meropenem/vaborbactam (95.1% susceptible by EUCAST and 93.9% susceptible by CLSI; MIC50 = 0.5 mg/L; MIC90 = 4 mg/L) and imipenem/relebactam (97% susceptible by EUCAST and 92.8% susceptible by CLSI; MIC50 = 0.25 mg/L; MIC90 = 0.5 mg/L) showed similar activity, followed by ceftazidime/avibactam (93.9% susceptible by both EUCAST and CLSI; MIC50 = 2 mg/L; MIC90 = 8 mg/L). Ten out of 13 (76.9%) KPC-Kp resistant to ceftazidime/avibactam carried a blaKPC variant including blaKPC-31, blaKPC-205, blaKPC-203 and blaKPC-93. Among KPC-Kp resistant to meropenem/vaborbactam and imipenem/relebactam, 90.9% (10/11) and 80% (4/5) harboured a WT carbapenemase (i.e. blaKPC-2 or blaKPC-3), respectively. All strains resistant to meropenem/vaborbactam and/or imipenem/relebactam carried truncated OmpK35 and/or mutated (ins135GD) OmpK36., Conclusions: New BLICs were shown to be the most widely active therapeutic option against KPC-Kp clinical isolates collected in Italy. Ceftazidime/avibactam resistance is mainly driven by the expression of KPC variants, whereas the loss of function of the OmpK35 and OmpK36 porins appears to play a key but not exclusive role in the development of meropenem/vaborbactam and/or imipenem/relebactam resistance., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Carbapenemase-producing Enterobacterales (CPEs) strains represent a serious threat to public health. The rapid diffusion of CPEs is of particular concern due to the limited effective treatments available against these multidrug resistant microorganisms. In this study, we characterized the complete genome sequence of Klebsiella pneumoniae strain BO714 coproducing KPC and OXA-181 carbapenemase conferring resistance to all β-lactam/β-lactamase inhibitor combinations (βL-βLICs) and siderophore cephalosporin cefiderocol (CFD). The genome of BO714 has a length of 5,876,068 bp with an average G + C content of 56.96% and a total of 5,878 open reading frames. The KPC-Kp strain BO714 was classified as ST512 and contained a circular chromosome of 5,348,787 bp and three different plasmids, respectively, of 363,560, 112,243, and 51,478 bp. Resistome analysis showed that BO714 harbored different β-lactamase genes including bla CMY-16 , bla OXA-10 , bla TEM-1 , bla SHV-11 , bla OXA181 , and a novel bla KPC-3 variant named bla KPC-125 . KPC-125 differed to KPC-3 by Asp to Ala at position 179 within the Ω-loop region. The genomic characterization of a K. pneumoniae cross-resistant to novel βL-βLICs and CFD improves knowledge regarding the emergence of novel traits of multidrug resistance in CPEs.

The spread of carbapenemase-producing Enterobacterales is a major public health concern worldwide, and methods for their prompt and reliable detection are highly demanded for therapeutic and hygiene control purposes. In this study, we evaluate the MBT STAR ® -Carba assay (Bruker Daltonik) to detect the carbapenemase production in clinical and surveillance isolates from plate cultures and directly from patient-derived positive blood cultures bottles. Overall, n  = 1,307 samples were analyzed ( n  = 900 plate cultures, and n  = 407 positive blood cultures, using the bacterial pellet obtained with the Sepsityper ® Kit; Bruker Daltonik), including n  = 793 carbapenemase producers ( n  = 579 Klebsiella pneumoniae carbapenemase, n  = 161 metallo-beta-lactamases, n  = 45 OXA-48, and eight isolates harboring two different enzymes), n  = 239 carbapenem-resistant noncarbapenemase producers, and n  = 275 carbapenem-susceptible strains. The STAR-Carba assay detected 657/661 (99.4%) carbapenemase producers from plate cultures, and 132/132 (100%) from positive blood cultures. Specificity resulted in 100% for carbapenem-susceptible strains, and 91.6% for carbapenem-resistant strains resulted negative for carbapenamase production with the routine methods used in this study. In this study, the MBT STAR-Carba assay proved to be a highly reliable method for the detection of carbapenemase-producing Enterobacterales , regardless of the enzyme family, and from both plate cultures and positive blood culture bottles.

Combination therapies are frequently used in the treatment of multidrug-resistant Klebsiella pneumoniae infection without consensus regarding which combination is the most effective. We compared bactericidal titres from sera collected from critically ill patients receiving meropenem plus tigecycline (n = 5), meropenem plus colistin (n = 5), or meropenem, colistin and tigecycline (n = 5) against K. pneumoniae isolates that included ESBL-producing (n = 7) and KPC-producing strains (n = 14) with varying sensitivity patterns to colistin and tigecycline. Meropenem concentrations (C min ) were measured in all samples by LC-MS/MS, and indexed to respective pathogen MICs to explore differences in patterns of bactericidal activity for two versus three drug combination regimens. All combination regimens achieved higher SBTs against ESBL (median reciprocal titre 128, IQR 32-256) versus KPC (4, IQR 2-32) strains. Sera from patients treated with meropenem-colistin yielded higher median SBTs (256, IQR 64-512) than either meropenem-tigecycline (32, IQR 8-256; P < 0.001). The addition of tigecycline was associated with a lower probability of achieving a reciprocal SBT above 8 when meropenem concentrations were below the MIC (P = 0.04). Although the clinical significance is unknown, sera from patients receiving tigecycline-based combination regimens produce lower serum bactericidal titres against ESBL or KPC-producing K. pneumoniae. SBTs may represent a useful complimentary endpoint for comparing pharmacodynamics of combinations regimens for MDR Enterobacteriaceae.

Here we report the complete nucleotide sequence of a 49.257-bp IncL/M conjugative plasmid (pRAY) carrying the blaOXA-48 gene collected from a Klebsiella pneumoniae clinical strain isolated in Italy. The genetic environment of pRAY plasmid revealed that the blaOXA-48 gene was located within a Tn1999.2 transposon. The pRAY plasmid differed from blaOXA-48-harboring IncL/M plasmids by genetic context and size. Comparative analysis demonstrated that pRAY plasmid lacked a region of ~15 kb carrying genes encoding proteins involved in pilus assembly and plasmid conjugative apparatus.

We evaluated a real-time single-peak (11.109-Da) detection assay based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae Our results demonstrated that the 11.109-Da peak was detected in 88.2% of the KPC producers. Analysis of blaKPC-producing K. pneumoniae showed that the gene encoding the 11.109-Da protein was commonly (97.8%) associated with the Tn4401a isoform., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We evaluated the analytical performance of a liquid chromatography-tandem mass spectrometry assay to detect carbapenemase activity in a group of carbapenemase-producing Enterobacteriaceae by meropenem hydrolysis. This one-hour method showed a sensitivity of 94% and a specificity of 100%, representing a rapid and reliable option compared to conventional phenotypic assays.

Objectives: Infections caused by Klebsiella pneumoniae (Kp) carbapenemase (KPC)-producing strains of Kp have become a significant threat in recent years. To assess their outcomes and identify risk factors for 14 day mortality, we conducted a 4 year (2010-13) retrospective cohort study in five large Italian teaching hospitals., Methods: The cohort included 661 adults with bloodstream infections (BSIs; n = 447) or non-bacteraemic infections (lower respiratory tract, intra-abdominal structure, urinary tract or other sites) caused by a KPC-Kp isolate. All had received ≥48 h of therapy (empirical and/or non-empirical) with at least one drug to which the isolate was susceptible., Results: Most deaths occurred within 2 weeks of infection onset (14 day mortality: 225/661, 34.1%). Logistic regression analysis identified BSI (OR, 2.09; 95% CI, 1.34-3.29), presentation with septic shock (OR, 2.45; 95% CI, 1.47-4.08), inadequate empirical antimicrobial therapy (OR, 1.48; 95% CI, 1.01-2.18), chronic renal failure (OR, 2.27; 95% CI, 1.44-3.58), high APACHE III score (OR, 1.05; 95% CI, 1.04-1.07) and colistin-resistant isolates (OR, 2.18; 95% CI, 1.37-3.46) as independent predictors of 14 day mortality. Combination therapy with at least two drugs displaying in vitro activity against the isolate was associated with lower mortality (OR, 0.52; 95% CI, 0.35-0.77), in particular in patients with BSIs, lung infections or high APACHE III scores and/or septic shock at infection onset. Combinations that included meropenem were associated with significantly higher survival rates when the KPC-Kp isolate had a meropenem MIC of ≤8 mg/L., Conclusions: KPC-Kp infections are associated with high mortality. Treatment with two or more drugs displaying activity against the isolate improves survival, mainly in patients who are critically ill., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Colistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceae producing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniae strains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrA and of pmrK, which is part of the pmrHFIJKLM operon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrB in KPB-2 restored colistin susceptibility and reduced the transcription of pmrA and pmrK to basal levels, while expression of PmrB(L82R) in KPB-1 did not alter colistin susceptibility or upregulate pmrA and pmrK expression, confirming the dominance of wild-type PmrB versus the PmrB(L82R) mutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Colistin is one of the few agents that retain activity against extensively drug-resistant strains of Klebsiella pneumoniae producing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of the mgrB gene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role of mgrB inactivation in acquired colistin resistance was confirmed by complementation experiments with wild-type mgrB, which restored colistin susceptibility in KKBO-4, and by construction of an mgrB deletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. Insertional mgrB inactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1 in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of the mgrB gene, upregulated transcription of phoP, phoQ, and pmrK (which is part of the pmrHFIJKLM operon) was detected. These findings confirmed the MgrB regulatory role in K. pneumoniae and were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of the pmrHFIJKLM operon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.

The spread of carbapenemases in Enterobacteriaceae is among the most important issues in the antimicrobial resistance. The rapid and recent diffusion of class A and B carbapenemases determined the need of specific diagnostic tests able to detect with high sensitivity this type of resistance and to discriminate between the different enzymes. The aim of this study was to test two carbapenemase detection assays, the Rosco Synergic and the Hyplex polymerase chain reaction-enzyme-linked immunosorbent assays for screening carbapenemase-producing Enterobacteriaceae. The phenotypic and genotypic tests were evaluated among 108 clinical isolates, including Klebsiella pneumoniae carbapenemase (KPC) (n=50) and metallo-β-lactamase- (MBL) (n=20), and AmpC- (n=10) producing Enterobacteriaceae. The commercial phenotypic assay showed a high sensitivity performance detecting all KPC and MBL producers, including New Delhi MBL 1 (NDM-1) strains. In addition, the Rosco Synergic assay was able to distinguish specifically between the different mechanisms that confer resistance to carbapenems in Enterobacteriaceae. We also demonstrated that the genotypic test was able to detect all the class A and B carbapenemases showing high sensitivity (100%) and specificity (98%) in a fast and reliable time. Based on these results, both the commercial phenotypic and the genotypic assays could be helpful as confirmatory and discriminatory tests for the detection of class A and class B carbapenemases.

Background: The spread of Klebsiella pneumoniae (Kp) strains that produce K. pneumoniae carbapenemases (KPCs) has become a significant problem, and treatment of infections caused by these pathogens is a major challenge for clinicians., Methods: In this multicenter retrospective cohort study, conducted in 3 large Italian teaching hospitals, we examined 125 patients with bloodstream infections (BSIs) caused by KPC-producing Kp isolates (KPC-Kp) diagnosed between 1 January 2010 and 30 June 2011. The outcome measured was death within 30 days of the first positive blood culture. Survivor and nonsurvivor subgroups were compared to identify predictors of mortality., Results: The overall 30-day mortality rate was 41.6%. A significantly higher rate was observed among patients treated with monotherapy (54.3% vs 34.1% in those who received combined drug therapy; P = .02). In logistic regression analysis, 30-day mortality was independently associated with septic shock at BSI onset (odds ratio [OR]: 7.17; 95% confidence interval [CI]: 1.65-31.03; P = .008); inadequate initial antimicrobial therapy (OR: 4.17; 95% CI: 1.61-10.76; P = .003); and high APACHE III scores (OR: 1.04; 95% CI: 1.02-1.07; P < .001). Postantibiogram therapy with a combination of tigecycline, colistin, and meropenem was associated with lower mortality (OR: 0.11; 95% CI: .02-.69; P = .01)., Conclusions: KPC-Kp BSIs are associated with high mortality. To improve survival, combined treatment with 2 or more drugs with in vitro activity against the isolate, especially those also including a carbapenem, may be more effective than active monotherapy.