Klebsiella Pneumoniae Carbapenemase-producing K. Pneumoniae Research Articles

In vitro mimicry of in vivo KPC mutations by ceftazidime-avibactam: phenotypes, mechanisms, genetic structure and kinetics of enzymatic hydrolysis

Ceftazidime-avibactam (CZA) is employed for the treatment of infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP). Resistance to CZA is frequently linked to point mutations in the bla KPC. We conducted in vitro simulations of in vivo bla KPC mutations using CZA. Four pre-therapy KPC-KP isolates (K1, K2, K3, and K4) were evaluated, all initially exhibited susceptibility to CZA and produced KPC-2. The crucial distinction was that following CZA treatment, the bla KPC-2 mutated in K1, K2, and K3, rendering them resistant to CZA, while K4 achieved microbiological clearance, and bla KPC-2 remained unaltered. The induction assay identified various bla KPC-2 variants, including bla KPC-25, bla KPC-127, bla KPC-100, bla KPC-128, bla KPC-137, bla KPC-138, bla KPC-144 and bla KPC-180. Our findings suggest that the resistance of KPC-KP to CZA primarily results from the emergence of KPC variants, complemented by increased bla KPC expression. A close correlation exists between avibactam concentration and the rate of increased CZA minimum Inhibitory concentration, as well as bla KPC mutation. Inadequate avibactam concentration is more likely to induce resistance in strains against CZA, there is also a higher likelihood of mutation in the bla KPC-2 and the optimal avibactam ratio remains to be determined. Simultaneously, we selected a bla KPC-33-producing K. pneumoniae strain (mutated from bla KPC-2) and induced it with imipenem and meropenem, respectively. The bla KPC-2 was detected during the process, indicating that the mutation is reversible. Clinical use of carbapenems to treat KPC variant strains increases the risk of infection, as the gene can mutate back to bla KPC-2, rendering the strain even more cross-resistant to carbapenems and CZA.

In vivo evolution of a Klebsiella pneumoniae capsule defect with wcaJ mutation promotes complement-mediated opsono-phagocytosis during recurrent infection.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) bloodstream infections are associated with high mortality. We studied clinical bloodstream KPC-Kp isolates to investigate mechanisms of resistance to complement, a key host defense against bloodstream infection. We tested growth of KPC-Kp isolates in human serum. In serial isolates from a single patient, we performed whole genome sequencing and tested for complement resistance and binding by mixing study, direct ELISA, flow cytometry, and electron microscopy. We utilized an isogenic deletion mutant in phagocytosis assays and an acute lung infection model. We found serum resistance in 16 of 59 (27%) KPC-Kp clinical bloodstream isolates. In five genetically-related bloodstream isolates from a single patient, we noted a loss-of-function mutation in the capsule biosynthesis gene, wcaJ. Disruption of wcaJ was associated with decreased polysaccharide capsule, resistance to complement-mediated killing, and surprisingly, increased binding of complement proteins. Furthermore, an isogenic wcaJ deletion mutant exhibited increased opsono-phagocytosis in vitro and impaired in vivo control in the lung after airspace macrophage depletion in mice. Loss of function in wcaJ led to increased complement resistance, complement binding, and opsono-phagocytosis, which may promote KPC-Kp persistence by enabling co-existence of increased bloodstream fitness and reduced tissue virulence.

In Vitro Evaluation of Increasing Avibactam Concentrations on Ceftazidime Activity against Ceftazidime/Avibactam-Susceptible and Resistant KPC-Producing Klebsiella pneumoniae Clinical Isolates.

The novel β-lactam/β-lactamase inhibitor combinations (βL-βLICs) are one of the last-line resources available against multidrug-resistant (MDR) Gram-negative bacteria. Among βL-βLICs, ceftazidime/avibactam (CAZ-AVI) demonstrated strong activity against carbapenem-resistant Enterobacterales (CRE). Avibactam was proven to restore bactericidal activity of ceftazidime, inhibiting both KPC and OXA-48-like β-lactamases. Despite this, emergence of CAZ-AVI-resistant strains in Enterobacterales has been reported. Herein, we evaluated the in vitro ceftazidime activity in the presence of increasing concentrations of avibactam by the broth microdilution method against CAZ-AVI-susceptible and resistant genome-characterized KPC-producing K. pneumoniae (KPC-Kp) clinical isolates. Strains expressing KPC and co-expressing KPC/OXA-181 carbapenemase were selected on the basis of the different phenotypic traits for novel βL-βLICs and cefiderocol. Notably, avibactam at 8 mg/L maintained the MIC of ceftazidime above the clinical breakpoint in 14 out of 15 (93%) KPC-Kp resistant to CAZ-AVI. A high concentration of avibactam (i.e., 64 mg/L) is required to observe a bactericidal activity of ceftazidime against 9 out of 15 (60%) CAZ-AVI-resistant isolates. In vitro evaluation showed that with the increase in the concentration of avibactam, ceftazidime showed high activity against CAZ-AVI-susceptible strains. High concentrations of avibactam in vivo are required for ceftazidime to be active against CAZ-AVI-resistant KPC-Kp.

Mutations in ompK36 differentially impact in vitro synergy of meropenem/vaborbactam and ceftazidime/avibactam in combination with other antibiotics against KPC-producing Klebsiella pneumoniae.

Ceftazidime/avibactam and meropenem/vaborbactam are preferred agents for Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) infections and are often used in combination with other agents. We aimed to characterize the synergy of combinations against KPC-Kp with varying ompK36 genotypes. KPC-Kp that harboured ompK36 WT, IS5 or glycine-aspartic acid duplication (GD) genotypes were selected. MICs were determined in triplicate. Synergy was assessed by time-kill assays for ceftazidime/avibactam and meropenem/vaborbactam in combination with colistin, gentamicin, tigecycline, meropenem or fosfomycin against 1 × 108 cfu/mL KPC-Kp. KPC-Kp harboured ompK36 WT (n = 5), IS5 (n = 5) or GD (n = 5); 11 were KPC-2 and 4 were KPC-3. All were susceptible to ceftazidime/avibactam and meropenem/vaborbactam. In time-kill analysis, ceftazidime/avibactam and meropenem/vaborbactam 1 × MIC exhibited mean 24 h log-kills of -2.01 and -0.84, respectively. Ceftazidime/avibactam was synergistic in combination with colistin independent of ompK36 genotype. Ceftazidime/avibactam combinations impacted by porin mutations (compared to WT) were meropenem (-5.18 versus -6.62 mean log-kill, P < 0.001) and fosfomycin (-3.98 versus -6.58, P = 0.058). Mean log-kills with meropenem/vaborbactam were greatest in combination with gentamicin (-5.36). In the presence of porin mutations, meropenem/vaborbactam killing activity was potentiated by the addition of colistin (-6.65 versus -0.70, P = 0.03) and fosfomycin (-3.12 versus 1.54, P = 0.003). Our results shed new light on the synergy of ceftazidime/avibactam and meropenem/vaborbactam combinations against KPC-Kp with or without porin mutations. Killing activity of ceftazidime/avibactam with other cell wall active agents was decreased against isolates with porin mutations. On the other hand, some meropenem/vaborbactam combinations demonstrated enhanced killing in the presence of porin mutations.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Epidemiology and In Vitro Activity of Ceftazidime/Avibactam, Meropenem/Vaborbactam and Imipenem/Relebactam against KPC-Producing K. pneumoniae Collected from Bacteremic Patients, 2018 to 2020.

The management of KPC-producing K. pneumoniae (KPC-Kp) in bloodstream infections (BSIs) represent a serious clinical challenge. In this study, the aim is to assess the incidence of resistance to novel β-lactams-β-lactamase inhibitor combinations (βL-βLICs), such as ceftazidime-avibactam (CAZ-AVI), meropenem-vaborbactam (MER-VAB) and imipenem-relebactam (IMI-REL), in KPC-Kp strains collected during a three-year period from patients with bacteremia. KPC-Kp strains resistant to βL-βLICs were selected for whole-genome sequencing. A total of 133 K. pneumoniae strains were isolated, and KPC-Kp strains were the most represented (87.2%). In 2018, resistance to CAZ-AVI and MER-VAB was 6.5% and 14.5%, respectively. In 2019, KPC-Kp resistance to CAZ-AVI and MER-VAB remained at low levels, with values of 12.9% and 3.2%, respectively. During 2020, CAZ-AVI resistance was detected in 2/23 of KPC-Kp strains (8.7%). IMI-REL was the most active βL-βLIC, inhibiting &gt;98% of the isolates, while CAZ-AVI and MER-VAB inhibited 87-93% and 85-97% of the KPC producers, respectively. Correlations between genotypic traits and resistance to βL-βLICs showed that KPC-Kp strains resistant to CAZ-AVI harbored a mutation within the blaKPC-3 gene, while all KPC-Kp strains resistant to CAZ-AVI, MER-VAB and/or IMI-REL carried the blaKPC-3 gene. Moreover, genetic analysis of porin genes showed that 14/16 of KPC-Kp resistant isolates possessed a truncated OmpK35 and glycine (G) and aspartic acid (D) insertions at positions 134-135 within OmpK36, whereas 2/16 displayed truncated OmpK35 and OmpK36 porins. Novel βL-βLICs are promising agents against KPC-Kp infections; however, the emergence of resistance to these agents highlights the need for continuous surveillance and application of enhanced antimicrobial stewardship.

Outbreak of KPC-producing Klebsiella pneumoniae at a Portuguese university hospital: Epidemiological characterization and containment measures.

KPC-producing K pneumoniae (KPC-Kp) is a public health problem with important clinical and epidemiological implications. We describe an outbreak of KPC-Kp at vascular surgery and neurosurgery wards in a central hospital in Porto, Portugal. A case of KPC-Kp was considered to be a patient positive for KPC-Kp with strong epidemiological plausibility of having acquired this microorganism in the affected wards and/or with genetic relationship ≥92% between KPC-Kp isolates. Active surveillance cultures (ASCs) and real-time polymerase chain reaction were used for the detection of carbapenemase genes through rectal swab in a selected population. Molecular analysis was performed using pulsed-field gel electrophoresis at the National Reference Laboratory. Patient risk factors were collected from the electronic medical record system. Information regarding outbreak containment strategy was collected from the Infection Control Unit records. Of the 16 cases, 11 (69%) were identified through active screening, representing 1.4% of the total 766 ASCs collected. The most frequent risk factors identified were previous admission (63%), antibiotic exposure in the past 6 months (50%), and immunodepression (44%). The length of stay until KPC-Kp detection was high (0-121 days, mean 35.6), as was the total length of stay (5-173 days, mean 56.6). Three patients (19%) were infected by KPC-Kp, 2 of whom died. One previously colonized patient died later because of KPC-Kp infection. Multifactorial strategy based on contact precautions (with patient and healthcare professional cohorts) and ASC, as well as Antibiotic Stewardship Program reinforcement, allowed to contain this KPC-Kp outbreak.

Novel insights related to the rise of KPC-producing Enterobacter cloacae complex strains within the nosocomial niche.

According to the World Health Organization, carbapenem-resistant Enterobacteriaceae (CRE) belong to the highest priority group for the development of new antibiotics. Argentina-WHONET data showed that Gram-negative resistance frequencies to imipenem have been increasing since 2010 mostly in two CRE bacteria: Klebsiella pneumoniae and Enterobacter cloacae Complex (ECC). This scenario is mirrored in our hospital. It is known that K. pneumoniae and the ECC coexist in the human body, but little is known about the outcome of these species producing KPC, and colonizing or infecting a patient. We aimed to contribute to the understanding of the rise of the ECC in Argentina, taking as a biological model both a patient colonized with two KPC-producing strains (one Enterobacter hormaechei and one K. pneumoniae) and in vitro competition assays with prevalent KPC-producing ECC (KPC-ECC) versus KPC-producing K. pneumoniae (KPC-Kp) high-risk clones from our institution. A KPC-producing E. hormaechei and later a KPC-Kp strain that colonized a patient shared an identical novel conjugative IncM1 plasmid harboring bla KPC-2. In addition, a total of 19 KPC-ECC and 58 KPC-Kp strains isolated from nosocomial infections revealed that high-risk clones KPC-ECC ST66 and ST78 as well as KPC-Kp ST11 and ST258 were prevalent and selected for competition assays. The competition assays with KCP-ECC ST45, ST66, and ST78 versus KPC-Kp ST11, ST18, and ST258 strains analyzed here showed no statistically significant difference. These assays evidenced that high-risk clones of KPC-ECC and KPC-Kp can coexist in the same hospital environment including the same patient, which explains from an ecological point of view that both species can exchange and share plasmids. These findings offer hints to explain the worldwide rise of KPC-ECC strains based on the ability of some pandemic clones to compete and occupy a certain niche. Taken together, the presence of the same new plasmid and the fitness results that showed that both strains can coexist within the same patient suggest that horizontal genetic transfer of bla KPC-2 within the patient cannot be ruled out. These findings highlight the constant interaction that these two species can keep in the hospital environment, which, in turn, can be related to the spread of KPC.

Community-acquired bacteraemia by Klebsiella pneumoniae producing KPC-3 and resistant to ceftazidime/avibactam

To describe the clinical and microbiological features of a case of community-acquired infection by KPC-producing K. pneumoniae (KPCKP) resistant to ceftazidime/avibactam (CAZ-AVI). Identification of microorganisms was performed with MALDI Biotyper CA System (BrukerDaltonics, Madrid, Spain). Antimicrobial susceptibility testing was performed using Sensitre EURGNCOL panels (Thermo Fisher Scientific, Madrid, Spain) and gradient strips (Etest, bioMérieux, Madrid, Spain) in the case of CAZ-AVI, using EUCAST breakpoints for interpretation. Whole genome sequencing of blood culture and rectal swab isolates was performed using the Illumina NovaSeq 6000 sequencing system, with 2×150-bp paired-end reads (Illumina, Inc.). Blood culture and rectal swab KPCKP isolates were resistant to carbapenems and to CAZ-AVI. The blood culture isolate showed susceptibility to trimethoprim-sulfamethoxazole (TMP-SMX), but the rectal swab culture isolate was resistant to this antibiotic. Both isolates belonged to clonal lineage ST512, harboured a single copy of blaKPC-3 gene, and showed 16 single nucleotide polymorphisms (SNP) between them and 38 SNPs with regard to the first KPC-3 producing K. pneumoniae isolated in our hospital in an initial outbreak in 2012. Genome-wide resistome analysis revealed the presence of a IncFIB(K) plasmid harbouring sul1 and dfrA12 genes only in the rectal swab culture isolate, which may explain its resistance to TMP-SMX. Resistance to ceftazidime-avibactam is an emerging nosocomial problem. This case shows that CAZ-AVI-resistant KPCKP strains may disseminate into the community and cause serious infections.

Increased blaKPC Copy Number and OmpK35 and OmpK36 Porins Disruption Mediated Resistance to Imipenem/Relebactam and Meropenem/Vaborbactam in a KPC-Producing Klebsiella pneumoniae Clinical Isolate.

Herein, we report the in vivo evolution of imipenem/relebactam-resistance in KPC-producing K. pneumoniae (KPC-Kp) isolated from a critically-ill patient treated with multiple combination therapies based on ceftazidime-avibactam or meropenem-vaborbactam. Imipenem/relebactam-resistance was associated to meropenem-vaborbactam cross-resistance and was due to truncated OmpK35 and OmpK36 porins and increased copy of blaKPC copy number. Genome analysis demonstrated that imipenem/relebactam-resistant KPC-Kp harbored a second copy of blaKPC-carrying Tn4401 in a ColRNAI plasmid as a consequence of a transposition event.

Reduced Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) colonization in a hematological-emergency setting during the coronavirus disease 2019 (COVID-19) pandemic.

Reduced Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) colonization in a hematological-emergency setting during the coronavirus disease 2019 (COVID-19) pandemic.

In vivo selection of KPC-94 and KPC-95 in Klebsiella pneumoniae isolates from patients treated with ceftazidime/avibactam

Ceftazidime/avibactam (CZA) is used to treat infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp). Resistance to CZA is commonly related to point mutations in the blaKPC gene. Here we describe the in vivo emergence of CZA resistance in clinical isolates of KPC-Kp from four patients treated with this combination therapy. Four pre-therapy and five post-therapy KPC-Kp isolates were examined. Antibiogram (microdilution and gradient strips) and whole-genome sequencing were performed. The role of KPC mutations was validated by cloning blaKPC genes into competent Escherichia coli. All KPC-Kp isolates recovered before treatment with CZA were susceptible to CZA and produced KPC-3. Five KPC-Kp isolates recovered after treatment were resistant to this combination. Three post-therapy isolates from two patients produced KPC-31 (D179Y mutation). Additionally, we identified the novel substitution LN169-170H (KPC-94) in one isolate, and the combination of two independently described mutations, D179Y and A172T (KPC-95), in another isolate. All KPC-Kp isolates belonged to sequence type 512 (ST512). All CZA-resistant isolates with blaKPC variants had restoration of carbapenem susceptibility. In conclusion, resistance to CZA was related to blaKPC mutations, including the new KPC-94 and KPC-95 alleles, which do not cause carbapenem resistance.

Rapid detection of KPC-producing Klebsiella pneumoniae in China based on MALDI-TOF MS

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) represent a serious threat to public health and their timely detection is essential for patient management and the prevention of nosocomial infections. Here, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to rapidly identify dominant KPC-Kp in China, by using an automated detection of a KPC-specific peak (at 4521 m/z) by a genetic algorithm using ClinProTools software. Whole-genome sequencing (WGS) was used to understand the genetic environment of the blaKPC-2 gene. In this study, we analyzed 235 K. pneumoniae Chinese clinical isolates, of which 175 (93 KPC-positive isolates and 82 KPC-negative isolates) isolates were used to build a model to select a KPC-specific peak, and another 60 isolates for external validation. In addition, all the spectra were visually inspected by the FlexAnalysis software to evaluate the accuracy of the automated detection. The results showed a 4521 m/z peak found in all blaKPC-2-positive isolates but absent in blaKPC-2-negative isolates. Interestingly, all KPC-Kp belonged to ST11, the dominant clone in China. WGS analysis of a representative isolate showed that the genetic environment of KPC-2 was IS26-ISKpn27-blaKPC-2-ΔISKpn6-Tn1721, similar to the KPC-2 genetic environment of ST11 KPC-Kp previously reported in China. Therefore, the 4521 m/z peak is closely related to ST11 KPC-Kp. In summary, we used MALDI-TOF MS to quickly detect KPC-Kp in the process of routine bacterial identification without increasing costs or requiring further knowledge, which has broad application prospects in drug resistance analysis and infection control.

Association between rectal colonisation by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae and mortality: a prospective, observational study

We evaluated the association of Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) rectal colonisation with crude mortality and whether this association is independent of the risk of KPC-Kp infection. This was a prospective cohort study of patients followed-up 90 days after a study of rectal colonisation. Cox regression was used to study the variables associated with crude mortality. Sensitivity analyses for 90-day crude mortality in different subcohorts were performed. A total of 1244 patients (1078 non-colonised and 166 colonised) were included. None of the non-colonised patients and 78 (47.0%) of the colonised patients developed KPC-Kp infection. The 90-day crude mortality was 18.0% (194/1078) in non-colonised patients and 41.6% (69/166) in colonised patients. Rectal colonisation was not associated with crude mortality [hazard ratio (HR)=1.03, 95% confidence interval (CI) 0.69-1.54; P=0.85] when the model was adjusted for severe KPC-Kp infection [INCREMENT-CPE score (ICS) > 7]. KPC-Kp infection with ICS > 7 was associated with an increased risk of all-cause mortality (HR=2.21, 95% CI 1.35-3.63; P=0.002). In the sensitivity analyses, KPC-Kp colonisation was not associated with mortality in any of the analysed subcohorts, including patients who did not develop KPC-Kp infection (HR=0.93, 95% CI 0.60-1.43; P=0.74). KPC-Kp rectal colonisation was not associated with crude mortality. Mortality increased when colonised patients developed severe KPC-Kp infection (ICS > 7). Rectal colonisation was a necessary although insufficient condition to die from a KPC-Kp infection.

Pharmacokinetic/pharmacodynamic modelling to evaluate the efficacy of various dosing regimens of ceftazidime/avibactam in patients with pneumonia caused by Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae: a multicentre study in northern China

The objective of this study was to evaluate the efficacy of different dosing regimens of ceftazidime/avibactam (CZA) in patients with Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) pulmonary infections. A total of 70 KPC-Kp strains were isolated from sputum and bronchoalveolar lavage samples of patients with pulmonary infections in three hospitals in northern China from April 2015 to October 2015. Monte Carlo simulation (MCS) was performed using population pharmacokinetic parameters of CZA combined with the minimum inhibitory concentration (MIC) distributions gained from antimicrobial susceptibility testing to predict the efficacy of different dosing regimens. Various CZA dosing regimens were modelled using MCS. The in vitro study showed potent activity of CZA against KPC-Kp strains with MIC50/90 values of 1/2 mg/L, with a susceptibility rate of 95.7%. The values of cumulative fraction of response (CFR) for bactericidal (50%fT>5×MIC) target were as follows: for patients with creatinine clearance (CLCr) >51 mL/min, the CFR was 96.01% for 2.5 g CZA every 12 h (q12h) and 97.14% for 2.5 g CZA every 8 h (q8h); and for patients with moderate renal impairment (CLCr >30 to ≤50 mL/min), the CFR was 95.75% for 1.25 g CZA q12h and 97.09% for 1.25 g CZA q8h. This study indicated that the recommended dose of CZA can provide adequate pharmacodynamic exposure for treating KPC-Kp pneumonia.

Efficacy of tigecycline alone or in combination for experimental infections by KPC carbapenemase-producing Klebsiella pneumoniae

Although in vitro data suggest that tigecycline is active against Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp), experimental and clinical data are limited. We studied the effect of tigecycline alone or in combination for experimental infections by KPC-Kp. A total of 540 male C57BL/6 mice were infected with three genetically diverse KPC-Kp isolates susceptible to tigecycline with meropenem minimum inhibitory concentrations (MICs) of 4, 16 and 256 μg/mL, respectively. Mice were randomly treated with water for injection, tigecycline, meropenem and colistin alone, and double or triple combinations of tigecycline, colistin and meropenem. Mouse survival was recorded for 14 days. In separate experiments, mice were sacrificed 6 h and 24 h after bacterial challenge for quantitative culture of tissues and serological analysis. Time-kill curves were performed. Tigecycline, colistin and meropenem concentrations were measured in tissues and serum by high-performance liquid chromatography (HPLC). Survival was significantly prolonged when mice were treated with tigecycline alone and tigecycline-containing regimens compared with control mice and mice treated with tigecycline-sparing regimens. Tigecycline-sparing regimens were active only against the isolate with a meropenem MIC of 4 μg/mL. Mortality was associated with progression to multiple organ failure. Tigecycline and tigecycline-containing regimens achieved a rapid decrease of bacterial loads both in tissues and in vitro. Tigecycline concentrations in tissues were negatively correlated with tissue bacterial load. Tigecycline alone or in combination with meropenem and/or colistin achieves effective treatment of experimental KPC-Kp infections irrespective of the meropenem MIC.

Epidemiology of Meropenem/Vaborbactam Resistance in KPC-Producing Klebsiella pneumoniae Causing Bloodstream Infections in Northern Italy, 2018.

Meropenem/Vaborbactam (MEM-VAB) is a novel carbapenem- β-lactamase inhibitor active against KPC-producing Enterobacteria. Herein, we evaluate the incidence of meropenem/vaborbactam-resistance among KPC-producing K. pneumoniae (KPC-Kp) bloodstream infection in a large Italian hospital. Meropenem/vaborbactam-resistance was found in 8% (n = 5) KPC-Kp, while 5% (n = 3) strains exhibited cross-resistance to ceftazidime/avibactam (CAZ-AVI). Genomic analysis revealed that meropenem/vaborbactam-resistance was associated with truncated OmpK35 and insertion of glycine and aspartic acid within OmpK36 at position 134–135 (GD134–135). Notably, no specific mutation was associated to cross-resistance. No specific antimicrobial treatment was related to favorable clinical outcomes, while cross-resistance was not associated to higher clinical and/or microbiological failures. Our study indicated that resistance to meropenem/vaborbactam was due to porins mutations and is associated with reduced susceptibility to both ceftazidime/avibactam and carbapenems.

In vitro Optimization of Ceftazidime/Avibactam for KPC-Producing Klebsiella pneumoniae.

Ceftazidime/avibactam is an important treatment option for infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp), however, resistance can emerge during treatment. The objective of the study was to define the ceftazidime/avibactam concentrations required to suppress bacterial regrowth in ceftazidime/avibactam susceptible isolates and identify active therapies against ceftazidime/avibactam-resistant KPC-Kp. Time-kill assays were performed against twelve ST258 KPC-Kp isolates that harbored blaKPC–2 or blaKPC–3. Nine KPC-Kp isolates (KPC-Kp 5A, 6A, 7A, 8A, 9A, 24A, 25A, 26A, and 27A) were susceptible to ceftazidime/avibactam, two (KPC-Kp 6B and 7B) were ceftazidime/avibactam resistant and meropenem susceptible, and one (KPC-Kp 1244) was resistant to both ceftazidime/avibactam and meropenem. Sequencing of the blaKPC genes revealed mutations in KPC-Kp 6B (D179Y substitution) and 7B (novel 21 base pair deletion) that both affected the Ω-loop encoding portion of the gene. Time-kill assays showed that against ceftazidime/avibactam-susceptible KPC-Kp, ceftazidime/avibactam concentrations ≥40/7.5 mg/L caused mean 5.42 log10CFU/mL killing and suppressed regrowth. However, regrowth occurred for some KPC-Kp isolates with a ceftazidime/avibactam concentration of 20/3.75 mg/L. Against ceftazidime/avibactam-resistant and meropenem-susceptible KPC-Kp 6B and 7B, bactericidal activity and synergy was observed for ceftazidime/avibactam in combination with meropenem ≤3.125 mg/L, while meropenem concentrations ≥50 mg/L were bactericidal as monotherapy. In contrast, clinically achievable concentrations of ceftazidime/avibactam were bactericidal against KPC-Kp 1244, which was ceftazidime/avibactam-resistant and meropenem-resistant due to outer membrane porin mutations and elevated blaKPC expression. Achieving high ceftazidime/avibactam concentrations may help to suppress bacterial regrowth in the presence of ceftazidime/avibactam. The optimal treatment approach for ceftazidime/avibactam-resistant KPC-Kp likely depends on the mechanism of resistance. Additional studies are warranted to confirm these findings.

Ceftazidime-Avibactam Use for Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae Infections: A Retrospective Observational Multicenter Study.

A growing body of observational evidence supports the value of ceftazidime-avibactam (CAZ-AVI) in managing infections caused by carbapenem-resistant Enterobacteriaceae. We retrospectively analyzed observational data on use and outcomes of CAZ-AVI therapy for infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-Kp) strains. Multivariate regression analysis was used to identify variables independently associated with 30-day mortality. Results were adjusted for propensity score for receipt of CAZ-AVI combination regimens versus CAZ-AVI monotherapy. The cohort comprised 577 adults with bloodstream infections (n = 391) or nonbacteremic infections involving mainly the urinary tract, lower respiratory tract, and intra-abdominal structures. All received treatment with CAZ-AVI alone (n = 165) or with ≥1 other active antimicrobials (n = 412). The all-cause mortality rate 30 days after infection onset was 25% (146/577). There was no significant difference in mortality between patients managed with CAZ-AVI alone and those treated with combination regimens (26.1% vs 25.0%, P = .79). In multivariate analysis, mortality was positively associated with presence at infection onset of septic shock (P = .002), neutropenia (P < .001), or an INCREMENT score ≥8 (P = .01); with lower respiratory tract infection (LRTI) (P = .04); and with CAZ-AVI dose adjustment for renal function (P = .01). Mortality was negatively associated with CAZ-AVI administration by prolonged infusion (P = .006). All associations remained significant after propensity score adjustment. CAZ-AVI is an important option for treating serious KPC-Kp infections, even when used alone. Further study is needed to explore the drug's seemingly more limited efficacy in LRTIs and potential survival benefits of prolonging CAZ-AVI infusions to ≥3 hours.

Risk Factors and Outcomes of Klebsiella pneumoniae Infection Before and After Allogeneic Hematopoietic Cell Transplantation.

Objectives: Klebsiella pneumoniae carbapenemase (KPC)–producing K. pneumoniae (KPC-Kp) emerge as a major healthcare concern worldwide. Despite the significance of infections before and after allogeneic hematopoietic cell transplantation (alloHCT), the burden of KP infections has not been extensively evaluated.Methods: We studied the incidence, risk factors, and outcomes of consecutive alloHCT recipients with Kp isolates before and after alloHCT.Results: Among 424 patients who underwent alloHCT in 2008–2018, we studied two groups: those with Kp isolates before (group 1, 52 patients) and those with Kp isolates after alloHCT (group 2, 66 patients). prE-transplant infections were associated with post-transplant infections (p = 0.010), despite secondary prophylaxis. KPC-Kp was isolated in 29% of group 1, and 80% of group 2. Both groups were characterized by a significant burden of moderate–severe acute graft- vs.-host disease (GVHD) [cumulative incidence (CI) of 44.5 and 61.9%, respectively] and severe chronic (CI of 56.7 and 61.9%). Kp infections and GVHD were independent predictive factors of treatment-related mortality (TRM) in both groups.Conclusions: Our study highlights the significant impact of Kp infections on TRM, with GVHD consisting an important underlying factor. As prophylactic measures did not improve rates of post-transplant infections, innovative interventions need to be further investigated to address this major healthcare concern.