KPC Enzymes Research Articles

National Cohort of Compassionate Use of Meropenem–Vaborbactam: No Benefit over Meropenem for Pseudomonas aeruginosa

Background: Meropenem–vaborbactam (MEM-VAB) is a novel carbapenem-beta-lactamase-inhibitor combination that demonstrates activity against carbapenem-resistant (CR) Gram-negative bacteria, and more specifically KPC-producers, since vaborbactam is an effective inhibitor of KPC enzymes in vitro. This study aimed to describe the initial uses and efficacy of MEM-VAB for compassionate treatment during the first 21 months following its early access in France. Method: A national multicenter retrospective study was conducted, including all patients who received at least one dose of MEM-VAB between 20 July 2020, and 5 April 2022. Clinical characteristics and outcomes were collected using a standardized questionnaire. The minimum inhibitory concentration (MIC) of antimicrobials, and complete genome sequencing of bacteria were performed when bacterial isolates were available. Results: Ultimately, 21 patients from 15 French hospitals were included in the study. The main indication for MEM-VAB treatment was respiratory tract infections (n = 9). The targeted bacteria included Pseudomonas aeruginosa (n = 12), Klebsiella pneumoniae (n = 3), Enterobacter spp (n = 3), Citrobacter freundii (n = 1), Escherichia coli (n = 1), and Burkholderia multivorans (n = 1). Overall, no significant advantage of vaborbactam over meropenem alone was observed across all strains of P. aeruginosa in terms of in vitro susceptibility. However, MEM-VAB demonstrated a notable impact, compared to carbapenem alone, on the MIC for the two KPC-3-producing K. pneumoniae and B. multivorans. Conclusions: MEM-VAB seems effective as a salvage treatment in compassionate use, but vaborbactam was shown to lack benefits compared to meropenem in treating P. aeruginosa-related infections. Therefore, it is crucial to compare meropenem to MEM-VAB MICs, particularly for P. aeruginosa, before prescribing MEM-VAB.

Antimicrobial Stewardship: A Game Changer in the Fight against Carbapenem-Resistant Enterobacteriaceae (CRE) among Children with Cancer

Abstract Background Carbapenem-resistant Enterobacteriaceae (CRE) is an emerging threat among pediatric cancer patients, with a high mortality rate. A previous study in our center reported that 254 patients with CRE BSI were diagnosed from (2013-2018) with mortality from CRE BSI being 30%. Previous colonization was reported in 35% of those patients. Time to start adequate antibiotics is an independent prognostic factor for patients with CRE BSI, (48%) of patients started active treatment after 48h from culture and associated with higher mortality when compared to patients who started treatment earlier. Our recommendations include antimicrobial stewardship for early detection through routine screening and timely adequate therapy that may impact the outcome for such high-risk patient groups. Methods This prospective study included all pediatric cancer patients with (CRE) bloodstream infections (BSI) at a children’s cancer hospital in Egypt (2020–2022) with adopting antimicrobial stewardship program goals by (1) applying routine screening by rectal swab for high-risk patients and starting active treatment for patients colonized by CRE. (2) Introduction of PCR-based method (Cepheid XpertCarba-R assay) is developed for detecting carbapenemase genes with the availability of results within one hour which can allow for rapid time to start active treatment in less than 48h. Results One hundred and fifty pediatric cancer patients with CRE BSI were identified; 83% had hematological malignancies, and 17% had solid tumors. Acute myeloid leukemia was the most common hematological malignancy (47%). The main clinical features for acquiring CRE-BSI were prolonged neutropenia > 7 days (62%), previous Carbapenem exposure (82%), Quinilone prophylaxis (44%), central venous catheter use (40%), previous colonization with a resistant pathogen (35%) and ICU admission within 90 days (18%). E. coli was the most common isolated pathogen (65%), followed by Klebsiella pneumoniae (31%). Documented site of infection was reported with pneumonia in (32%), typhlitis in 15% skin soft tissue infection (57%), CNS infection (18%), and urinary tract infection in (10%). Time to start active antibiotics in less than 48h was reported in 89% with clinical cure reported in 71% and microbiological clearance was reported in in less than 7 days in 70% of patients. The need for ICU admission as sepsis related to CRE was reported in 29/150 (19%). The Gentopyic profile for CRE in our study reported that class B (69%) with NDM reported in 98/150 (65%) and VIM 6/150 (4%) was the most common carbanamases followed by Oxa-48 in 89/150(59%) of patients while KPC enzyme reported only in 6/150(4%). Day 30 mortality was reported in 23/150(15%). Upon multivariate analysis, patients with Klebsiella pneumoniae BSI, NDM genotype carbapenemases, CRE with aminoglycosides resistance or associated with pneumonia, and CRE patients who developed septic shock with the need for ICU admission were predictors of poor outcome. Conclusion CRE-BSI is a major threat among pediatric cancer patients in limited-resource countries and is considered a barrier against better outcomes. Antimicrobial stewardship through routine screening for colonization detection and Rapid diagnosis by using gene expert with rapid time to start adequate active antibiotics treatment is associated with improvement of outcome and decreasing mortality. Table 1: Comparison between retrospective study and prospective study (after adopting antimicrobial stewardship goals)

Pharmacodynamics of taniborbactam in combination with cefepime studied in an in vitro model of infection

ObjectivesTo define the in vitro pharmacodynamics of taniborbactam against Enterobacterales with CTXM-15, KPC, AmpC, and OXA-48 β-lactamases. MethodsAn in vitro pharmacokinetic model was used to simulate serum concentrations associated with cefepime 2G by 1 h infusion 8 h. Taniborbactam was given in exposure ranging and fractionation simulations. Reduction in viable count at 24 h (Δ 24) was the primary end point and four strains were used: Escherichia coli expressing CTXM-15 or AmpC and Klebsiella pneumoniae expressing KPC or OXA-48 enzymes. ResultsTaniborbactam was administered as continuous infusions; ≥4 log kill was attained with taniborbactam concentrations of ≥0.01 mg/L against CTXM-15 E. coli, ≥0.5 mg/L against KPC- and OXA-48 K. pneumoniae, and ≥4 mg/L against AmpC E. coli. Analyses were conducted to determine the pharmacokinetic/dynamic driver for each strain. For E. coli (CTXM-15) and E. coli (AmpC), area under the concentration-time curve (AUC) was best related to change in viable count (R20.74 and 0.72, respectively). For K. pneumoniae (KPC) AUC and T > 0.25 mg/L were equally related to bacterial clearance (R20.72 for both), and for K. pneumoniae (OXA-48) T > 0.25 mg/L was the best predictor (R20.94). The taniborbactam AUC range to produce a 1-log10 reduction in viable count was 4.4–11.2 mg·h/L. Analysis of data from all strains indicated T > MIC divided by 4 was best related to change in viable count; however, curve fit was poor R2 < 0.49. ConclusionsTaniborbactam was effective in combination with cefepime in producing bacterial clearance for B lactam resistant Enterobacterales. The primary pharmacodynamic driver was AUC or time > threshold, both being closely related to antibacterial effect.

Analysis of the molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae in a hospital in Hunan Province

To examine the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) and investigate the horizontal transmission of blaKPC and blaNDM genes for the prevention and treatment of CRKP. A total of 49 clinically isolated CRKP strains were retrospectively analyzed from January to December 2022 at The First Hospital of Hunan University of Chinese Medicine. Phenotypic screening was performed using modified carbapenem inactivation assay (mCIM) and EDTA-carbapenem inactivation assay (eCIM). Polymerase chain reaction (PCR) was utilized to identify carbapenem resistance genes, β-lactamase resistance genes, and virulence genes, while multi-locus sequence analysis (MLST) was employed to assess the homology of CRKP strains. Conjugation experiments were conducted to infer the horizontal transmission mechanism of blaKPC and blaNDM genes. The results showed that the study included 49 CRKP strains, with 44 carrying blaKPC and 8 carrying blaNDM, Three strains were identified as blaKPC+blaNDM-CRKP. In this study, 28 out of 49 CRKP strains (57.2%) were found to carry virulence genes. Additionally, one CRKP strain tested positive in the string test and was found to carry both Aerobactin and rmpA virulence genes. MLST results revealed a total of 5 ST types, with ST11 being predominant (41/49, 83.7%). Successful conjugation was observed in all 3 blaKPC-CRKP strains, while only 1 out of 3 blaNDM-CRKP strains showed successful conjugation. The transconjugant exhibited significantly reduced susceptibility to imipenem and cephalosporin antibiotics. In conclusion, the resistance mechanism of CRKP in this study is primarily attributed to the production of KPC enzymes, along with the presence of multiple β-lactamase resistance genes. Additionally, there is a local prevalence of hv-CRKP and blaKPC+blaNDM-CRKP. blaKPC and blaNDM can be horizontally transmitted through plasmids, with varying efficiency among different strains.

A large-scale surveillance revealed that KPC variants mediated ceftazidime-avibactam resistance in clinically isolated Klebsiella pneumoniae.

To monitor the resistance rate and gain a deeper understanding of the resistance mechanisms, we conducted over a 2-year surveillance focusing on the Klebsiella pneumoniae associated with the clinical usage of ceftazidime-avibactam (CZA) in a teaching hospital. A total of 4,641 K. pneumoniae isolates were screened to identify the CZA resistance through antimicrobial susceptibility testing. Comprehensive analyses, including homology analysis, conjugation experiments, clone assays, and whole genome sequencing, were furtherly performed on the CZA-resistant strains. In total, four CZA-resistant K. pneumoniae (CZA-R-Kp) strains were separated from four patients, in which three of them received CZA treatment during the hospitalization, accounting for a 4% (3/75) resistance development rate of K. pneumoniae under CZA stress. All CZA-R-Kp isolates were found to possess variants of blaKPC-2. The identified mutations included blaKPC-33, blaKPC-86, and a novel variant designated as blaKPC-129, all of which were located in the Ω loop of the KPC enzyme. These mutations were found to impact the amino acid sequence and spatial structure of the enzyme's active center, consequently affecting KPC carbapenemase activity. This study underscores the importance of active surveillance to monitor the emergence of resistance to CZA, highlighting the need for ongoing research to develop effective strategies for combating antimicrobial resistance. Understanding the mechanisms behind resistance is crucial in maintaining the efficacy of CZA, a vital tool in the battle against multidrug-resistant infections.IMPORTANCEAs an effective drug for the treatment of carbapenem-resistant Klebsiella pneumoniae, ceftazidime-avibactam (CZA) began to develop resistance in recent years and showed an increasing trend. In order to effectively monitor the resistance rate of CZA and understand its resistance mechanism, we monitored K. pneumoniae for more than 2 years to find CZA-resistant strains. Through comprehensive analysis of the selected CZA-resistant strains, it was found that all the CZA-resistant strains had mutation, which could affect the activity of KPC carbapenemase. This study highlights the importance of proactive surveillance to monitor the emergence of CZA resistance, which highlights the need for ongoing research to develop effective strategies to combat antimicrobial resistance. Understanding the mechanisms behind resistance is critical to maintaining the effectiveness of CZA, an important tool in the fight against multidrug-resistant infections.

Antimicrobial resistance trends among Klebsiella pneumoniae associated with urinary tract infections in Crete, Greece, 2017-2022.

Klebsiella pneumoniae is one of the most prevalent bacteria causing urinary tract infections (UTIs). Its increasing resistance to a wide array of antibiotics limits available treatment options. This study investigated the characteristics and trends of antimicrobial resistance in K. pneumoniae isolated from UTIs in Crete, Greece, during 2017 and 2022. Among the 11,946 Enterobacteriaceae isolated from urine specimens, a total of 1,771 K. pneumoniae isolates were identified (14.8%), with an isolation frequency secondary to Escherichia coli (66.3%). K. pneumoniae isolates increased over the years, with a peak in the year 2022. Higher resistance rates were detected in ciprofloxacin (41%), trimethoprim/sulfamethoxazole (TMP/SMX) (38.1%) and nitrofurantoin (33.9%). Resistance to ciprofloxacin, amoxicillin/clavulanic acid, tigecycline, and TMP/SMX significantly increased from 33.7%, 24%, 6%, and 33.1%, respectively, over the years 2017-2019, to 47.8%, 34.2%, 14.3% and 42.8%, respectively, over the period 2020-2022. ESBL production and carbapenem resistance were decreased by 2.2% and 3.7%, respectively, over the two three-year periods (2017-2019 and 2020-2022). Among the 278 carbapenem-resistant K.pneumoniae (CRKP) isolates, 164 (59%), 66 (23.7%), 18 (6.5%) and 16 (5.8%) were positive for KPC, NDM, VIM and OXA-48 enzymes, respectively. Only 14 (5%) isolates harboured two carbapenemase genes, namely 10 (3.6%) both blaNDM and blaVIM, and 4 (1.4%) both blaKPC and blaNDM. Females, inpatients and the elderly were more frequently affected by CRKP. The frequency of multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were 32.6% and 7.7%, respectively. Continuous surveillance of local microbial prevalence and monitoring of antimicrobial resistance patterns provide critical information to guide the empiric therapy for UTIs and control the spread of MDR bacteria.

Comparative study of phenotypic-based detection assays for carbapenemases in Acinetobacter baumannii

BackgroundAcinetobacter baumannii is a serious health concern worldwide, causing high mortality rates and limited medical therapy options. Carbapenem resistance is a significant problem in Acinetobacter baumannii isolates. The synthesis of acquired carbapenemases, such as oxacillinases, IMP, NDM, VIM, and KPC enzymes, causes carbapenem resistance. MethodsA total of 106 non-repetitive, Acinetobacter baumannii isolates were collected from four major hospitals in Bahrain including 78 carbapenem-resistant Acinetobacter baumannii (CRAB), and 28 carbapenem-susceptible Acinetobacter baumannii (CSAB) isolates. Three phenotypic tests were investigated in this study: including CARBA NP, modified carbapenem inactivation method (mCIM)/EDTA-CIM (eCIM), and modified Hodge test (MHT). ResultsCARBA NP was positive in 50 tested CRAB isolates (100%), and the sensitivity was 100%. The MHT was positive in 73/106 isolates (68.8%), while the sensitivity and specificity of the MHT were 77.6% and 100%. Moreover, only 38/106 (35.8%) isolates were positive for mCIM/eCIM. The sensitivity and specificity of mCIM were 40.4% and 100%. ConclusionCARBA NP was ideal for phenotypic detection of carbapenemase production, followed by MHT. The m/eCIM demonstrated a lower detection rate in CRAB. Consequently, combining tests would be more accurate. The mCIM/eCIM can easily distinguish between MBLs and serine-carbapenemases due to the frequent co-production of these enzymes in A. baumannii. In hospital setups where molecular characterization tests are not available, CARBA NP seems to be an alternative test in combination with MHT or mCIM/eCIM.

Synergistic Activity of Temocillin and Fosfomycin Combination against KPC-Producing Klebsiella pneumoniae Clinical Isolates.

Infections caused by KPC-producing K. pneumoniae continue to pose a significant clinical challenge due to their emerging resistance to new antimicrobials. We investigated the association between two drugs whose roles have been repurposed against multidrug-resistant bacteria: fosfomycin and temocillin. Temocillin exhibits unusual stability against KPC enzymes, while fosfomycin acts as a potent "synergizer". We conducted in vitro antimicrobial activity studies on 100 clinical isolates of KPC-producing K. pneumoniae using a combination of fosfomycin and temocillin. The results demonstrated synergistic activity in 91% of the isolates. Subsequently, we assessed the effect on Galleria mellonella larvae using five genetically different KPC-Kp isolates. The addition of fosfomycin to temocillin increased larvae survival from 73 to 97% (+Δ 32%; isolate 1), from 93 to 100% (+Δ 7%; isolate 2), from 63 to 86% (+Δ 36%; isolate 3), from 63 to 90% (+Δ 42%; isolate 4), and from 93 to 97% (+Δ 4%; isolate 10). Among the temocillin-resistant KPC-producing K. pneumoniae isolates (24 isolates), the addition of fosfomycin reduced temocillin MIC values below the resistance breakpoint in all isolates except one. Temocillin combined with fosfomycin emerges as a promising combination against KPC-producing K. pneumoniae, warranting further clinical evaluation.

Antibiotic resistance and epidemiological characteristics of polymyxin-resistant Klebsiella pneumoniae.

The emergence of polymyxin-resistant Klebsiella pneumoniae (KPN) in clinical settings necessitates an analysis of its antibiotic resistance characteristics, epidemiological features, and risk factors for its development. This study aims to provide insights for the prevention and control of polymyxin-resistant KPN infections. Thirty clinical isolates of polymyxin-resistant KPN were collected from the Third Xiangya Hospital of Central South University. Their antibiotic resistance profiles were analyzed. The presence of carbapenemase KPC, OXA-48, VIM, IMP, and NDM was detected using colloidal gold immunochromatography. Hypervirulent KPN was initially screened using the string test. Biofilm formation capacity was assessed using crystal violet staining. Combination drug susceptibility tests (polymyxin B with meropenem, tigecycline, cefoperazone/sulbactam) were conducted using the checkerboard method. Polymyxin-related resistance genes were detected by PCR. Multi-locus sequence typing (MLST) was performed for genotyping and phylogenetic tree construction. The study also involved collecting data from carbapenem-resistant (CR)-KPN polymyxin-resistant strains (23 strains, experimental group) and CR-KPN polymyxin-sensitive strains (57 strains, control group) to analyze potential risk factors for polymyxin-resistant KPN infection through univariate analysis and multivariate Logistic regression. The induction of resistance by continuous exposure to polymyxin B and colistin E was also tested. Among the 30 polymyxin-resistant KPN isolates, 28 were CR-KPN, all producing KPC enzyme. Four isolates were positive in the string test. Most isolates showed strong biofilm formation capabilities. Combination therapy showed additive or synergistic effects. All isolates carried the pmrA and phoP genes, while no mcr-1 or mcr-2 genes were detected. MLST results indicated that ST11 was the predominant type. The phylogenetic tree suggested that polymyxin-resistant KPN had not caused a hospital outbreak in the institution. The use of two or more different classes of antibiotics and the use of polymyxin were identified as independent risk factors for the development of polymyxin-resistant strains. Continuous use of polymyxin induced drug resistance. Polymyxin-resistant KPN is resistant to nearly all commonly used antibiotics, making polymyxin-based combination therapy a viable option. No plasmid-mediated polymyxin-resistant KPN has been isolated in the hospital. Polymyxin can induce resistance in KPN, highlighting the need for rational antibiotic use in clinical settings to delay the emergence of resistance.

Detection of KPC directly from positive blood cultures by MALDI-TOF: From research to the clinical microbiology laboratory routine

Bloodstream infections (BSI) caused by carbapenem-resistant Gram-negative bacilli (CR-GNB) are a subject of major clinical concern, mainly those associated with carbapenemase-producing isolates. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed to detect specific β-lactamases, including KPC. We aimed to detect KPC enzyme directly from positive blood cultures using MALDI-TOF MS. Overall, 146 clinical Gram-negative bacilli (46 CR-GNB) recovered from consecutive blood cultures were evaluated. Proteins were extracted using formic acid, isopropyl alcohol, and water and spotted onto a steel target plate using the double-layer sinapinic acid method. The relative ions intensity ≥120 arbitrary units (a.u.) of a peak close to 28,700 m/z indicated the presence of KPC. The results were compared to HRM-qPCR methodology. This specific peak was observed in 11/14 blood bottles with blaKPC positive isolates (78.6% sensitivity), with 3 false-positive results (97.7% specificity). Analysis from colonies reached identical sensitivity (78.6%), but higher specificity (100%). The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid. It's excellent specificity indicates that positive results are consistently associated with the presence of a KPC producer in positive blood culture.

High-risk clones of carbapenem resistant Klebsiella pneumoniae recovered from pediatric patients in Southern Brazil.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) exhibit high mortality rates in pediatric patients and usually belong to international high-risk clones. This study aimed to investigate the molecular epidemiology and carbapenem resistance mechanisms of K.pneumoniae isolates recovered from pediatric patients, and correlate them with phenotypical data. Twenty-five CRKP isolates were identified, and antimicrobial susceptibility was assessed using broth microdilution. Carbapenemase production and β-lactamase genes were detected by phenotypic and genotypic tests. Multilocus sequence typing was performed to differentiate the strains and whole-genome sequencing was assessed to characterize a new sequence type. Admission to the intensive care unit and the use of catheters were significantly positive correlates of CRKP infection, and the mortality rate was 36%. Almost all isolates showed multidrug-resistant phenotype, and most frequent resistant gene was blaKPC. We observed the dissemination of ST307 and clones belonging to CG258, which are considered high risk. In pediatric patients, these clones present with high genomic plasticity, favoring adaptation of the KPC and NDM enzymes to healthcare environments.

KPC-2 allelic variants in Klebsiella pneumoniae isolates resistant to ceftazidime-avibactam from Argentina: blaKPC-80, blaKPC-81, blaKPC-96 and blaKPC-97.

The emergence of ceftazidime-avibactam (CZA) resistance poses a significant threat to the efficacy of this life-saving therapy against carbapenem-resistant bacteria, particularly Klebsiella pneumoniae-producing KPC enzymes. This study investigates four clinical isolates exhibiting resistance to CZA, revealing novel allelic variants of the key resistance gene, blaKPC-2. The mutations identified in hotspots surrounding the active site of KPC, such as K269_D270insPNK, del_I173, Y241N and V277_I278insNSEAV, prove the adaptability of these pathogens. Intriguingly, low-level resistance to imipenem and disruptions in porin genes were observed, emphasizing the complexity of the resistance mechanisms. Interestingly, three of four isolates belonged to clonal complex 11. This research not only sheds light on the clinical significance of CZA resistance but also shows the urgency for comprehensive surveillance and molecular studies to inform effective antimicrobial treatment strategies in the face of evolving bacterial resistance.

In Vitro Activity of "Old" and "New" Antimicrobials against the Klebsiella pneumoniae Complex.

The Klebsiella pneumoniae complex is a commonly isolated bacteria in human infections. These opportunistic pathogens pose a serious threat to public health due to their potential transmission to the human population. Resistance to carbapenems is a significant antimicrobial resistance mechanism, leading to limited therapeutic options. Therefore, the aim of this study was to evaluate the in vitro activity of fosfomycin, colistin, ceftazidime-avibactam, and meropenem-vaborbactam against multidrug-resistant K. pneumoniae complex strains. This study involved 160 strains of Gram-negative rods, comprising 138 K. pneumoniae and 22 K. variicola. The minimal inhibitory concentration of fosfomycin was estimated using the agar dilution method, and for colistin, the microdilution method was employed. Susceptibility to ceftazidime-avibactam and meropenem-vaborbactam was determined using the gradient strip method. All analyzed K. pneumoniae complex isolates produced extended-spectrum β-lactamases, and 60.0% exhibited carbapenemases. The majority of the analyzed strains were susceptible to fosfomycin and colistin (62.5%). Among pandrug-resistant K. pneumoniae complex isolates, the highest susceptibility was observed with colistin (43.9%). Fosfomycin demonstrated good activity against ESβLs- and VIM-positive isolates from this complex. Colistin also exhibited satisfactory in vitro activity against VIM- and KPC-positive isolates from the K. pneumoniae complex. Ceftazidime-avibactam displayed good activity against K. pneumoniae complex strains producing ESβLs, KPC, and OXA enzymes. Additionally, meropenem-vaborbactam showed satisfactory in vitro activity against ESβLs- and KPC-positive isolates from this complex.

Inoculum effect of CTX-M-15, OXA-48, and KPC-2 producing Klebsiella pneumoniae on meropenem and ceftazidime-avibactam efficacy.

The inoculum effect, characterized by diminished antibacterial activity at high bacterial inocula, is studied in the context of beta-lactam and beta-lactamase inhibitor combinations against beta-lactamase-producing Enterobacterales. The inhibition of ESBL + OXA-48 and KPC enzymes, in combination with ceftazidime, demonstrates encouraging results. In this study, 20 Klebsiella pneumoniae isolates were tested with different inocula (1-5 × 105 and 1-5 × 107cfu/ml) using broth microdilution methods. The inoculum effect was observed in meropenem against OXA-48 + CTX-M-15- and KPC-2-producing isolates but not with ceftazidime/avibactam. Notably, meropenem exhibited inoculum effect against carbapenemase-producing strains, whereas ceftazidime-avibactam remained effective. We conclude that ceftazidime-avibactam is recommended for high-inoculum infections.

Antimicrobial resistance of clinical isolates of Klebsiella pneumoniae and Escherichia coli in Russian hospitals: results of a multicenter epidemiological study

Objective. To study the prevalence and mechanisms of antibiotic resistance, including carbapenemase production, in clinical isolates of Klebsiella pneumoniae and Escherichia coli isolated in different regions of Russia as part of the sentinel multicenter surveillance study in 2020–2021, and to explore the population structure of K. pneumoniae and the impact of “high-risk clones” on antibiotic resistance. Materials and Methods. Consecutive, non-duplicate isolates of K. pneumoniae (n = 2503) and E. coli (n = 2055) isolated from various specimens (blood, cerebrospinal fluid, respiratory samples, urine, wound secretions, etc.) of hospitalized patients with clinical signs of infection in 55 hospitals of 29 cities of Russia were studied. Species identification of isolates was performed by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS). Antibiotic susceptibilities were determined by serial broth microdilution or, in the case of fosfomycin, by agar dilution method, and results were interpreted according to EUCAST v13 MIC breakpoints. Carbapenemase production was determined phenotypically by carbapenem inactivation method (CIM), the presence of genes of the most common serine carbapenemases (KPC, OXA-48) and metallo-β-lactamases (VIM, IMP, NDM) was determined by real-time PCR. K. pneumoniae clinical isolates were genotyped and assigned to the known clonal complexes (CC) and sequence types (ST) using SNP typing and multilocus sequencing typing (MLST) methods. K- and O-serotypes, acquired resistance and virulence genes, and plasmids carrying these genes were characterized using whole-genome sequencing of selected isolates (n = 215). Results. The resistance rates of nosocomial/community-acquired isolates of K. pneumoniae were as follows: amoxicillin-clavulanate – 88.63⁄57.99%, piperacillin-tazobactam – 82.92⁄45.49%, cefotaxime – 87.74⁄56.97%, ceftazidime – 84.76⁄53.07%, cefepime – 81.43⁄49.18%, aztreonam – 1.63⁄53.28%, ceftazidime-avibactam – 30, 88⁄9.22%, ceftolozan-tazobactam – 70.06/31.35%, ertapenem – 72.10⁄28.69%, meropenem – 49.60⁄15.16%, imipenem – 44.54⁄13.73%, gentamicin – 60.82⁄30.33%, amikacin – 42.06/17.21%, ciprofloxacin – 85.10⁄49.39%; trimethoprimsulfamethoxazole – 74.38⁄48.16%, colistin – 5.96⁄2.25%. The resistance of nosocomial/outpatient isolates of E. coli were: ampicillin – 84.93⁄67.67%, amoxicillin-clavulanate – 57.37⁄39.73%, piperacillin-tazobactam – 19.48⁄8.70%, cefotaxime – 63.83⁄34.19%, ceftazidime – 45.32⁄20.34%, cefepime – 35.95⁄16.61%, aztreonam – 51.78⁄26.11%, ceftazidime-avibactam – 5.71⁄0.80%, ceftolozane-tazobactam – 11, 95⁄2.22%, ertapenem – 8.18⁄1.42%, meropenem – 5.17⁄0.53%, imipenem – 4.95⁄0.36%, gentamicin – 24.54⁄13.68%, amikacin – 5.49⁄1.42%, ciprofloxacin – 54, 14⁄32.50%, trimethoprim-sulfamethoxazole – 52.21⁄38.54%, fosfomycin – 2.48⁄1.43%, colistin – 1.60⁄1.07%, tigecycline – 6.35⁄3.11%. The frequency of carbapenemase production among K. pneumoniae nosocomial isolates was 65.32% (OXA-48 – 40.75%, NDM – 30.28%, KPC – 8.74%, OXA-48 + NDM – 10.62%, OXA-48 + KPC – 2.98%, NDM + KPC – 0.45%, OXA-48 + NDM + KPC – 0.20%). More than 70% of nosocomial isolates of K. pneumoniae belonged to only 7 major genetic lineages known as “high-risk international clones”: CC395 – 37.40%, CC23 – 9.59%, CC307 – 8.64%, CC147 – 7.61%, CC15 – 2.95%, CC258 – 2.92%, and CC11 – 2.41%. The population of community-acquired K. pneumoniae was characterized by significantly greater genetic diversity (Simpson diversity index: D = 0.919; 95% CI: 0.904 to 0.933) compared with the population of nosocomial strains (Simpson diversity index: D = 0.815; 95% CI: 0.802 to 0.827). Strains of the “hypervirulent” genetic lineage of K. pneumoniae CC23 were more common in community-acquired infections. Conclusions. The extremely high frequency of resistance to cephalosporins in K. pneumoniae (&gt; 80%) and E. coli (&gt; 60%), as well as the high frequency of combined resistance to aminoglycosides and fluoroquinolones precludes their empirical use for the treatment of serious nosocomial infections caused by these pathogens. K. pneumoniae shows a rapid increase in resistance to carbapenems, mainly due to the spread of carbapenemases of three major groups: OXA-48, NDM and KPC. The overall increase in the frequency of carbapenemase production is accompanied by the growing diversity of carbapenemases, the increasing prevalence of strains producing NDM and KPC enzymes and those co-producing multiple carbapenemases simultaneously. In community-acquired infections, the high prevalence of resistance to cephalosporins in E. coli (&gt; 30%) and K. pneumoniae (&gt; 50%) remains the most important problem.

A critical role of outer membrane vesicles in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae

BackgroundThis study aimed to illustrate the status of carbapenem-resistant Enterobacterales (CRE) infections in a Chinese tertiary hospital and to investigate the role of outer membrane vesicles (OMVs) in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP).MethodsThe data of CRE infections was collected from laboratory records, and the CRE isolates from two distinct periods (2015/07 to 2017/07 and 2020/04 to 2021/04) were enrolled to detect the carbapenemase genes by polymerase chain reaction (PCR). Multilocus sequence typing (MLST) was used to analyze the molecular characterization of CRKP. The conjugation assay was performed to verify the transmission of the antibiotic resistance plasmid. The OMVs of CRKP were isolated with a method combining an electrophoretic technique with a 300 kDa cut-off dialysis bag. The protein components in CRKP OMVs were analyzed by liquid chromatography tandem-mass spectrometry (LC–MS/MS), and the meropenem-hydrolyzing bioactivity of KPC in CRKP OMVs was determined with different treatments in vitro.ResultsA total of 178 CRE isolates, including 100 isolates from 2015/07 to 2017/07 and 78 isolates from 2020/04 to 2021/04, were collected for the detection of carbapenemase genes. We found that the carbapenemase gene blaKPC was the most prevalent, followed by blaNDM. By MLST, we found that sequence type (ST) 11 CRKP (96.1%) was the leading type during 2015/07 to 2017/07 and that the ST15 CRKP increased to 46.2% in the late period of 2020/04 to 2021/04. The diameters of Klebsiella pneumoniae OMVs ranged from 100 to 200 nm, and by proteomics analysis the most proteins from OMVs belonged to the “enzyme” group. The KPC enzyme was found in the OMVs from CRKP, and the OMVs could protect inside KPC from proteinase K digestion. Moreover, the KPC enzymes within OMVs, which could be released after Triton X-100 treatment, could hydrolyze meropenem.ConclusionsCRE has increasingly caused infections in hospitals, and blaKPC-positive CRKP infections have constituted a major proportion of infections in the past decade. The OMVs play a critical role in antibiotic resistance in CRKP.

Evaluation of thymol effect on antimicrobial susceptibility and biofilm production in clinical Enterobacter spp. producing ESBLs and KPC enzymes

ABSTRACT Background Multidrug-resistant Enterobacter strains that harbour Klebsiella pneumoniaecarbapenemase and Extended Spectrum β-Lactamases genes are a therapeuticchallenge in hospitals that have made infection control and antibiotic therapydifficult and use of natural compounds such as thymol is interesting in thetreatment of infections. Methods Sixty-seven Enterobacter spp. isolates were obtained from patients admittedto hospitals in Hamadan and Tehran. Antimicrobial susceptibility, ESBLs andKPC genes detection and biofilm production of isolates were measured. Findings The results showed that the highest and lowest antibiotic resistance wererelated to ceftazidime (82.1%) and tigecycline (3%), respectively. Also, 79.1% of the isolates were MDR. The highestpresence of genes was related to TEM (100%), SHV (82%), CTX-M(77%) and KPC (8.96%) genes. The MICand MBIC of thymol were obtained 8-31 μg/ml and16-62 μg/ml, respectively. Conclusion Use of thymol, as an aid in antibiotic therapy is recommended.

Aromatic Diboronic Acids as Effective KPC/AmpC Inhibitors.

Over 30 compounds, including para-, meta-, and ortho-phenylenediboronic acids, ortho-substituted phenylboronic acids, benzenetriboronic acids, di- and triboronated thiophenes, and pyridine derivatives were investigated as potential β-lactamase inhibitors. The highest activity against KPC-type carbapenemases was found for ortho-phenylenediboronic acid 3a, which at the concentration of 8/4 mg/L reduced carbapenems' MICs up to 16/8-fold, respectively. Checkerboard assays revealed strong synergy between carbapenems and 3a with the fractional inhibitory concentrations indices of 0.1-0.32. The nitrocefin hydrolysis test and the whole cell assay with E. coli DH5α transformant carrying blaKPC-3 proved KPC enzyme being its molecular target. para-Phenylenediboronic acids efficiently potentiated carbapenems against KPC-producers and ceftazidime against AmpC-producers, whereas meta-phenylenediboronic acids enhanced only ceftazidime activity against the latter ones. Finally, the statistical analysis confirmed that ortho-phenylenediboronic acids act synergistically with carbapenems significantly stronger than other groups. Since the obtained phenylenediboronic compounds are not toxic to MRC-5 human fibroblasts at the tested concentrations, they can be considered promising scaffolds for the future development of novel KPC/AmpC inhibitors. The complexation of KPC-2 with the most representative isomeric phenylenediboronic acids 1a, 2a, and 3a was modeled by quantum mechanics/molecular mechanics calculations. Compound 3a reached the most effective configuration enabling covalent binding to the catalytic Ser70 residue.

The CARBA-MAP study: national mapping of carbapenemases in Spain (2014-2018).

Infections caused by carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa, including isolates producing acquired carbapenemases, constitute a prevalent health problem worldwide. The primary objective of this study was to determine the distribution of the different carbapenemases among carbapenemase-producing Enterobacterales (CPE, specifically Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae complex, and Klebsiella aerogenes) and carbapenemase-producing P. aeruginosa (CPPA) in Spain from January 2014 to December 2018. A national, retrospective, cross-sectional multicenter study was performed. The study included the first isolate per patient and year obtained from clinical samples and obtained for diagnosis of infection in hospitalized patients. A structured questionnaire was completed by the participating centers using the REDCap platform, and results were analyzed using IBM SPSS Statistics 29.0.0. A total of 2,704 carbapenemase-producing microorganisms were included, for which the type of carbapenemase was determined in 2692 cases: 2280 CPE (84.7%) and 412 CPPA (15.3%), most often using molecular methods and immunochromatographic assays. Globally, the most frequent types of carbapenemase in Enterobacterales and P. aeruginosa were OXA-48-like, alone or in combination with other enzymes (1,523 cases, 66.8%) and VIM (365 cases, 88.6%), respectively. Among Enterobacterales, carbapenemase-producing K. pneumoniae was reported in 1821 cases (79.9%), followed by E. cloacae complex in 334 cases (14.6%). In Enterobacterales, KPC is mainly present in the South and South-East regions of Spain and OXA-48-like in the rest of the country. Regarding P. aeruginosa, VIM is widely distributed all over the country. Globally, an increasing percentage of OXA-48-like enzymes was observed from 2014 to 2017. KPC enzymes were more frequent in 2017-2018 compared to 2014-2016. Data from this study help to understand the situation and evolution of the main species of CPE and CPPA in Spain, with practical implications for control and optimal treatment of infections caused by these multi-drug resistant organisms.

Evaluation of the Ability to Form Biofilms in KPC-Producing and ESBL-Producing Klebsiella pneumoniae Isolated from Clinical Samples.

The appearance of Klebsiella pneumoniae strains producing extended-spectrum β-lactamase (ESBL), and carbapenemase (KPC) has turned into a significant public health issue. ESBL- and KPC-producing K. pneumoniae's ability to form biofilms is a significant concern as it can promote the spread of antibiotic resistance and prolong infections in healthcare facilities. A total of 45 K. pneumoniae strains were isolated from human infections. Antibiograms were performed for 17 antibiotics, ESBL production was tested by Etest ESBL PM/PML, a rapid test was used to detect KPC carbapenemases, and resistance genes were detected by PCR. Biofilm production was detected by the microtiter plate method. A total of 73% of multidrug resistance was found, with the highest resistance rates to ampicillin, trimethoprim-sulfamethoxazole, cefotaxime, amoxicillin-clavulanic acid, and aztreonam. Simultaneously, the most effective antibiotics were tetracycline and amikacin. blaCTX-M, blaTEM, blaSHV, aac(3)-II, aadA1, tetA, cmlA, catA, gyrA, gyrB, parC, sul1, sul2, sul3, blaKPC, blaOXA, and blaPER genes were detected. Biofilm production showed that 80% of K. pneumoniae strains were biofilm producers. Most ESBL- and KPC-producing isolates were weak biofilm producers (40.0% and 60.0%, respectively). There was no correlation between the ability to form stronger biofilms and the presence of ESBL and KPC enzymes in K. pneumoniae isolates.