Genomic characterization of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) strains circulating in three university hospitals in Northern Italy over three years.

OBJECTIVES: Genomic surveillance of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is crucial for virulence, drug-resistance monitoring, and outbreak containment. METHODS: Genomic analysis on 87 KPC-Kp strains isolated from 3 Northern Italy hospitals in 2019-2021 was performed by whole genome sequencing (WGS), to characterize resistome, virulome, and mobilome, and to assess potential associations with phenotype resistance and clinical presentation. Maximum Likelihood and Minimum Spanning Trees were used to determine strain correlations and identify potential transmission clusters. RESULTS: Overall, 15 different STs were found; the predominant ones included ST307 (35, 40.2%), ST512/1519 (15, 17.2%), ST20 (12, 13.8%), and ST101 (7, 8.1%). 33 (37.9%) KPC-Kp strains were noticed to be in five transmission clusters (median number of isolates in each cluster: 5 [3-10]), four of them characterized by intra-hospital transmission. All 87 strains harbored Tn4401a transposon, carrying blaKPC-3 (48, 55.2%), blaKPC-2 (38, 43.7%), and in one case (1.2%) blaKPC-33, the latter gene conferred resistance to ceftazidime/avibactam (CZA). Thirty strains (34.5%) harbored porin mutations; of them, 7 (8.1%) carried multiple Tn4401a copies. These strains were characterized by significantly higher CZA minimum inhibitory concentration compared with strains with no porin mutations or single Tn4401a copy, respectively, even if they did not overcome the resistance breakpoint of 8 ug/mL. Median 2 (IQR:1-2) virulence factors per strain were detected. The lowest number was observed in ST20 compared to the other STs (p<0.001). While ST307 was associated with infection events, a trend associated with colonization events could be observed for ST20. CONCLUSIONS: Integration of genomic, resistance score, and clinical data allowed us to define a relative diversification of KPC-Kp in Northern Italy between 2019 and 2021, characterized by few large transmission chains and rare inter-hospital transmission. Our results also provided initial evidence of correlation between KPC-Kp genomic signatures and higher MIC levels to some antimicrobial agents or colonization/infection status, once again underlining WGS's importance in bacterial surveillance.

Prevalence of Infections and Antimicrobial Resistance of ESKAPE Group Bacteria Isolated from Patients Admitted to the Intensive Care Unit of a County Emergency Hospital in Romania.

The ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella Pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) is a group of bacteria very difficult to treat due to their high ability to acquire resistance to antibiotics and are the main cause of nosocomial infections worldwide, posing a threat to global public health. Nosocomial infections with MDR bacteria are found mainly in Intensive Care Units, due to the multitude of maneuvers and invasive medical devices used, the prolonged antibiotic treatments, the serious general condition of these critical patients, and the prolonged duration of hospitalization. MATERIALS AND METHODS: During a period of one year, from January 2023 to December 2023, this cross-sectional study was conducted on patients diagnosed with sepsis admitted to the Intensive Care Unit of the Sibiu County Emergency Clinical Hospital. Samples taken were tracheal aspirate, catheter tip, pharyngeal exudate, wound secretion, urine culture, blood culture, and peritoneal fluid. RESULTS: The most common bacteria isolated from patients admitted to our Intensive Care Unit was Klebsiella pneumoniae, followed by Acinetobacter baumanii and Pseudomonas aeruginosa. Gram-positive cocci (Enterococcus faecium and Staphilococcus aureus) were rarely isolated. Most of the bacteria isolated were MDR bacteria. CONCLUSIONS: The rise of antibiotic and antimicrobial resistance among strains in the nosocomial environment and especially in Intensive Care Units raises serious concerns about limited treatment options.

TiO2 Nanocomposite Coatings and Inactivation of Carbapenemase-Producing Klebsiella Pneumoniae Biofilm-Opportunities and Challenges.

The worldwide increase of multidrug-resistant Gram-negative bacteria is a global threat. The emergence and global spread of Klebsiella pneumoniae carbapenemase- (KPC-) producing Klebsiella pneumoniae represent a particular concern. This pathogen has increased resistance and abilities to persist in human reservoirs, in hospital environments, on medical devices, and to generate biofilms. Mortality related to this microorganism is high among immunosuppressed oncological patients and those with multiple hospitalizations and an extended stay in intensive care. There is a severe threat posed by the ability of biofilms to grow and resist antibiotics. Various nanotechnology-based strategies have been studied and developed to prevent and combat serious health problems caused by biofilm infections. The aim of this review was to evaluate the implications of nanotechnology in eradicating biofilms with KPC-producing Klebsiella pneumoniae, one of the bacteria most frequently associated with nosocomial infections in intensive care units, including in our department, and to highlight studies presenting the potential applicability of TiO2 nanocomposite materials in hospital practice. We also described the frequency of the presence of bacterial biofilms on medical surfaces, devices, and equipment. TiO2 nanocomposite coatings are one of the best long-term options for antimicrobial efficacy due to their biocompatibility, stability, corrosion resistance, and low cost; they find their applicability in hospital practice due to their critical antimicrobial role for surfaces and orthopedic and dental implants. The International Agency for Research on Cancer has recently classified titanium dioxide nanoparticles (TiO2 NPs) as possibly carcinogenic. Currently, there is an interest in the ecological, non-toxic synthesis of TiO2 nanoparticles via biological methods. Biogenic, non-toxic nanoparticles have remarkable properties due to their biocompatibility, stability, and size. Few studies have mentioned the use of nanoparticle-coated surfaces as antibiofilm agents. A literature review was performed to identify publications related to KPC-producing Klebsiella pneumoniae biofilms and antimicrobial TiO2 photocatalytic nanocomposite coatings. There are few reviews on the antibacterial and antibiofilm applications of TiO2 photocatalytic nanocomposite coatings. TiO2 nanoparticles demonstrated marked antibiofilm activity, but being nano in size, these nanoparticles can penetrate cell membranes and may initiate cellular toxicity and genotoxicity. Biogenic TiO2 nanoparticles obtained via green, ecological technology have less applicability but are actively investigated.

Clinically relevant sequence types of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae detected in Finnish wastewater in 2021-2022.

BACKGROUND: Antimicrobial resistance (AMR) is a critical threat to human health. Escherichia coli and Klebsiella pneumoniae are clinically the most important species associated with AMR and are the most common carbapenemase-producing (CP) Enterobacterales detected in human specimens in Finland. Wastewater surveillance has emerged as a potential approach for population-level surveillance of AMR, as wastewater could offer a reflection from a larger population with one sample and minimal recognized ethical issues. In this study, we investigated the potential of wastewater surveillance to detect CP E. coli and K. pneumoniae strains similar to those detected in human specimens. METHODS: Altogether, 89 composite samples of untreated community wastewater were collected from 10 wastewater treatment plants across Finland in 2021-2022. CP E. coli and K. pneumoniae were isolated using selective culture media and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed using disk diffusion test and broth microdilution method, and a subset of isolates was characterized using whole-genome sequencing. RESULTS: CP E. coli was detected in 26 (29.2%) and K. pneumoniae in 25 (28.1%) samples. Among E. coli, the most common sequence type (ST) was ST410 (n = 7/26, 26.9%), while ST359 (n = 4/25, 16.0%) predominated among K. pneumoniae. Globally successful STs were detected in both E. coli (ST410, ST1284, ST167, and ST405) and K. pneumoniae (ST512, ST101, and ST307). K. pneumoniae carbapenemases (KPC) were the most common carbapenemases in both E. coli (n = 11/26, 42.3%) and K. pneumoniae (n = 13/25, 52.0%), yet also other carbapenemases, such as blaNDM-5, blaOXA-48, and blaOXA-181, were detected. We detected isolates harboring similar ST and enzyme type combinations previously linked to clusters in Finland, such as E. coli ST410 with blaKPC-2 and K. pneumoniae ST512 with blaKPC-3. CONCLUSIONS: Our study highlights the presence of clinically relevant strains of CP E. coli and K. pneumoniae in community wastewater. The results indicate that wastewater surveillance could serve as a monitoring tool for CP Enterobacterales. However, the specificity and sensitivity of the methods should be improved, and technologies, like advanced sequencing methods, should be utilized to distinguish data with public health relevance, harness the full potential of wastewater surveillance, and implement the data in public health surveillance.

In Vitro Activity of Cefiderocol, Cefepime-Zidebactam, and ß-Lactam Combinations Versus Other Antibiotic Classes Against Various Sequence Types of Clinically Isolated Carbapenemase-Producing Klebsiella pneumoniae.

Aim: This study aimed to establish the in vitro efficacy and susceptibility profiles of new ß-lactam antibiotics against clinically isolated carbapenemase-producing Klebsiella pneumoniae (CPKP) strains. Materials and Methods: A total of 117 nonduplicated CPKP isolates were tested against cefiderocol, cefepime-zidebactam, ceftazidime-avibactam, tigecycline, and other 20 antibiotics by broth microdilution. The carbapenemase genes were identified using PCR and sequencing, while multilocus sequence typing established the bacterial strains. Results: Three significant sequence types (STs), including ST147, ST16, and ST11, were shown to be the dominant STs, which occupied ∼90% of the tested population. Three carbapenemase genes, blaNDM-1, blaOXA-181, and blaOXA-232, were detected. The blaNDM-1 was found in ST147 and ST16 but not in ST11, while the blaOXA-232 was not detected in ST147. The majority of ST16 isolates contained both blaNDM-1 and blaOXA-232, which was not seen in other strains. Cefiderocol, cefepime-zidebactam, and tigecycline were the most active agents against CPKP. Both MIC50 and MIC90 of these three antibiotics remained within the susceptible categories, while nearly all other antibiotics were in the resistant levels. However, in ST11, which carried only blaOXA genes without blaNDM-1, ceftazidime-avibactam was effective with the MIC90 at 2 µg/mL. In addition, amikacin was shown to have good activity in ST11. In contrast, gentamicin was active in only ST16 and ST147. Conclusions: This study is the first report that demonstrates the prevalence of CPKP, distribution of strains, resistant genes, and antimicrobial susceptibility profiles in northern Thailand. These data would contribute to appropriate individual treatment and the selection of infection control strategies.

Direct prediction of carbapenem-resistant, carbapenemase-producing, and colistin-resistant Klebsiella pneumoniae isolates from routine MALDI-TOF mass spectra using machine learning and outcome evaluation.

The objective of this study was to develop a rapid prediction method for carbapenem-resistant Klebsiella pneumoniae (CRKP) and colistin-resistant K. pneumoniae (ColRKP) based on routine MALDI-TOF mass spectrometry (MS) results in order to formulate a suitable and rapid treatment strategy. A total of 830 CRKP and 1462 carbapenem-susceptible K. pneumoniae (CSKP) isolates were collected; 54 ColRKP isolates and 1592 colistin-intermediate K. pneumoniae (ColIKP) isolates were also included. Routine MALDI-TOF MS, antimicrobial susceptibility testing, NG-Test CARBA 5, and resistance gene detection were followed by machine learning (ML). Using the ML model, the accuracy and area under the curve for differentiating CRKP and CSKP were 0.8869 and 0.9551, respectively, and those for ColRKP and ColIKP were 0.8361 and 0.8447, respectively. The most important MS features of CRKP and ColRKP were m/z 4520-4529 and m/z 4170-4179, respectively. Of the CRKP isolates, MS m/z 4520-4529 was a potential biomarker for distinguishing KPC from OXA, NDM, IMP, and VIM. Of the 34 patients who received preliminary CRKP ML prediction results (by texting), 24 (70.6%) were confirmed to have CRKP infection. The mortality rate was lower in patients who received antibiotic regimen adjustment based on the preliminary ML prediction (4/14, 28.6%). In conclusion, the proposed model can provide rapid results for differentiating CRKP and CSKP, as well as ColRKP and ColIKP. The combination of ML-based CRKP with preliminary reporting of results can help physicians alter the regimen approximately 24 h earlier, resulting in improved survival of patients with timely antibiotic intervention.

In vitro activity of newer ß-lactam/ß-lactamase inhibitor combinations, cefiderocol, plazomicin and comparators against carbapenemase-producing Klebsiella pneumoniae isolates.

Infections by carbapenem-resistant Klebsiella pneumoniae (CRKP) remain one of the greatest healthcare threats associated with significant morbidity and mortality. New antimicrobials were recently developed to address this threat. We assessed the epidemiology of carbapenemase-producing K. pneumoniae (CPKP) isolates recovered in a Greek university hospital during 2021, and their susceptibilities to old and newer antimicrobials. Minimum inhibitory concentrations (MICs) were determined by the MIC Test Strip method, except for cefiderocol (CFDC) and colistin that were evaluated by the broth microdilution method. A total of 110 CPKP strains were isolated, with KPC-producers being the most prevalent (64.6%). Among the agents tested, plazomicin (PL) displayed the highest activity against all the isolates (MIC50/MIC90, 0.5/1.5 µg/ml), followed by tigecycline (MIC50/MIC90, 1.5/4 µg/ml). All KPC-producing K. pneumoniae were susceptible to ceftazidime-avibactam (CAZ/AVI) and meropenem-vaborbactam (M/V) and 97.2% of them to imipenem-relebactam (I/R). Among the MBL-producing isolates, PL and CFDC exhibited the highest activity.

Infection prevention and control policies in hospitals and prevalence of highly resistant microorganisms: an international comparative study.

BACKGROUND: There are differences in infection prevention and control (IPC) policies to prevent transmission of highly resistant microorganisms (HRMO). The aim of this study is to give an overview of the IPC policy of six European hospitals and their HRMO prevalence, to compare the IPC policies of these hospitals with international guidelines, and to investigate the hospitals' adherence to their own IPC policy. METHODS: The participating hospitals were located in Salzburg (Austria), Vienna (Austria), Kayseri (Turkey), Piraeus (Greece), Rome (Italy) and Rotterdam (The Netherlands). Data were collected via an online survey. Questions were aimed at prevalence rates in the years 2014, 2015, 2016 of carbapenemase-producing Klebsiella pneumoniae (CPK), carbapenemase-producing Pseudomonas aeruginosa (CPPA), vancomycin-resistant Enterococcus faecium (VRE) and hospitals' IPC policies of 2017. Implemented IPC measures (i.e. with a self-reported adherence of > 90%) were counted (26 points maximal). RESULTS: The self-reported prevalence of CPK per year was low in the Austrian and Dutch hospitals and high in the Turkish and Greek hospitals. CPPA was highly prevalent in the Turkish hospital only, while the prevalence of VRE in four hospitals, except the Austrian hospitals which reported lower prevalence numbers, was more evenly distributed. The Dutch hospital had implemented the most IPC measures (n = 21), the Turkish and Greek hospitals the least (n = 14 and 7, respectively). CONCLUSION: Hospitals with the highest self-reported prevalence of CPK and CPPA reported the least implemented IPC measures. Also, hospitals with a higher prevalence often reported a lower adherence to own IPC policy.

Comparison of performance of MALDI-TOF MS and MLST for biotyping carbapenemase-producing Klebsiella pneumoniae sequence types ST11 and ST101 isolates.

INTRODUCTION: The rapid identification and detection of carbapenemase-producing Klebsiella pneumoniae (CPKP) isolates is crucial to ascertain outbreaks, as well as to limit their spread. The current reference method for this purpose is multilocus sequence typing (MLST), which is laborious and expensive. Consequently, alternative typing methods are gaining attention, such as Matrix-Assisted Laser Desorption Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). METHODS: This study sought to analyze MALDI-TOF MS as a typing method using 44 CPKP isolates that were well characterized by MLST. The most common types of samples from which these pathogens were isolated were skin and soft tissues (32%) and urine (29%). Half of the CPKP isolates were from hospitalized patients. Two approaches were followed for the analysis of the mass peak data obtained by MALDI-TOF MS. The first using all peaks obtained and the second using a selection of 21 characteristic peaks. RESULTS: The selection of 21 characteristic peaks showed greater discrimination power for ST11 and ST101. Principal component analysis (PCA) indicated that this dataset could be efficiently grouped with lineal classifiers. A Support Vector Machine (SVM) was chosen for this purpose after checking its capacity to classify bacterial strains on the basis of MALDI-TOF MS information. CONCLUSION: SVM was able to discriminate between ST11 and ST101 with high accuracy. In conclusion, our results reveal MALDI-TOF MS as a promising alternative technique for typing of CPKP isolates.

Comparison of Two Distinct Subpopulations of Klebsiella pneumoniae ST16 Co-Occurring in a Single Patient.

The higher resistance rate to ceftazidime-avibactam (CZA) is mainly related to carbapenem resistance, especially New Delhi metallo-ß-lactamase (NDM). The CZA-susceptible Klebsiella pneumoniae (K191663) and the later CZA-resistant isolates (K191724, K191725, K191773) co-producing NDM-4 and OXA-181 were obtained from the same hospitalized patient returning from Vietnam. Our study aims to elucidate the diversity of K. pneumoniae ST16 through comparative analysis of whole-genome sequencing (WGS) data and identify the potential evolution of plasmids by sequencing longitudinal clinical isolates during antibiotic treatment. Firstly, multilocus sequence typing analysis and phylogenic analysis suggested that these strains belong to the two lineages of K. pneumoniae ST16. Surprisingly, the CZA-resistant strains were closely related to K. pneumoniae ST16 described in South Korea, instead of the blaNDM-4- or blaOXA-181-carrying ST16 reported in Vietnam. Secondly, blaNDM-4, blaTEM-1B, and rmtB co-existed on a self-conjugative IncFII(Yp)-like plasmid, which played a significant role in CZA resistance. It could transfer into the recipient Escherichia coli J53 at high frequency, indicating the risk of mobile carbapenemases. In addition, the loss of 12-kbp fragment occurred in blaNDM-4-positive isolate (K191773), which was likely caused by insertion sequence-mediated homologous recombination. Last but not least, as a repressor of acrAB operon system, acrR was truncated by a frameshift mutation in K191663. Thus, our study provided baseline information for monitoring the occurrence and development of bacterial resistance. IMPORTANCE As a leading health care-acquired infection pathogen, Klebsiella pneumoniae is threatening a large number of inpatients due to its diverse antibiotic resistance and virulence factors. Heretofore, with a growing number of reports about the coexistence of several carbapenemases in carbapenem-resistant K. pneumoniae (CRKP), epidemiologic surveillance has been strengthened. Nevertheless, the nosocomial outbreaks by CRKP ST16 are gradually increasing worldwide. Our study provides a deeper insight into the diversification of clinical isolates of CRKP ST16 in China. In addition, the comparison analysis of resistant plasmids may reveal the transmission of carbapenemase-encoding genes. Furthermore, our study also highlights the importance of longitudinal specimen collection and continuous monitoring during the treatment, which play a crucial role in understanding the development of antibiotic resistance and the evolution of resistance plasmids.

Comparison of performance of MALDI-TOF MS and MLST for biotyping carbapenemase-producing Klebsiella pneumoniae sequence types ST11 and ST101 isolates / Estudio comparativo de la utilidad de MALDI-TOF MS y MLST para el biotipado de Klebsiella pneumoniae ST11 y ST101 productoras de carbapenemasas

IntroductionThe rapid identification and detection of carbapenemase-producing Klebsiella pneumoniae (CPKP) isolates is crucial to ascertain outbreaks, as well as to limit their spread. The current reference method for this purpose is multilocus sequence typing (MLST), which is laborious and expensive. Consequently, alternative typing methods are gaining attention, such as Matrix-Assisted Laser Desorption Ionization–Time Of Flight Mass Spectrometry (MALDI-TOF MS).MethodsThis study sought to analyze MALDI-TOF MS as a typing method using 44 CPKP isolates that were well characterized by MLST. The most common types of samples from which these pathogens were isolated were skin and soft tissues (32%) and urine (29%). Half of the CPKP isolates were from hospitalized patients. Two approaches were followed for the analysis of the mass peak data obtained by MALDI-TOF MS. The first using all peaks obtained and the second using a selection of 21 characteristic peaks.ResultsThe selection of 21 characteristic peaks showed greater discrimination power for ST11 and ST101. Principal component analysis (PCA) indicated that this dataset could be efficiently grouped with lineal classifiers. A Support Vector Machine (SVM) was chosen for this purpose after checking its capacity to classify bacterial strains on the basis of MALDI-TOF MS information.ConclusionSVM was able to discriminate between ST11 and ST101 with high accuracy. In conclusion, our results reveal MALDI-TOF MS as a promising alternative technique for typing of CPKP isolates.

IntroducciónLa rápida identificación y detección de los aislados de Klebsiella pneumoniae productores de carbapenemasas (CPKP) es crucial para identificar brotes e impedir la propagación de los aislados resistentes. El método de referencia para este propósito es el multilocus sequencing typing (MLST), que es un técnica laboriosa y cara, por lo que se buscan métodos de tipado alternativos que pueden desempeñar la misma función con menor esfuerzo. Entre las posibles técnicas se encuentra la espectrometría de masas de tiempo de vuelo MALDI-TOF.MétodosEste estudio se han utilizado el sistema MALDI-TOF MS para tipar 44 aislamientos de CPKP previamente caracterizados por MLST. Las muestras clínicas de las que proceden los aislados son principalmente piel y tejidos blandos (32%) y orina (29%). La mitad de los aislamientos de CPKP procedían de pacientes ingresados. El análisis los datos obtenidos por MALDI-TOF MS se realizó con 2 enfoques diferentes, el primero usando todos los picos obtenidos y el segundo usando una selección de picos.ResultadosLa selección de 21 picos característicos ofreció un mayor poder de discriminación entre ST11 y ST101. El análisis de componentes principales (PCA) indicó que este conjunto de datos podría agruparse eficientemente con clasificadores lineales. Para realizar este agrupamiento se escogió el algoritmo support vector machine (SVM, máquinas de vectores de soporte) para este propósito después de verificar su capacidad para clasificar las cepas bacterianas en base a la información de MALDI-TOF MS.ConclusiónSVM pudo discriminar entre ST11 y ST101 con alta precisión. En conclusión, nuestros resultados revelan MALDI-TOF MS puede ser una técnica alternativa para el tipificación de aislamientos de CPKP.

Association between Timing of Colonization and Risk of Developing Klebsiella pneumoniae Carbapenemase-Producing K. pneumoniae Infection in Hospitalized Patients.

Colonization by KPC-producing Klebsiella pneumoniae (KPC-Kp) is associated with the risk of developing KPC-Kp infection. The impact of the time elapsed since a patient becomes colonized on this risk is not well known. An observational, prospective, longitudinal cohort study of colonized patients undergoing active rectal culture screening to rule out KPC-Kp colonization (July 2012 to November 2017). Patients with a positive culture at inclusion (colonized at start of follow-up) and those with a negative culture at inclusion who became colonized within 90 days (colonized during follow-up) were included in the analysis. CART analysis was used to dichotomize variables according to their association with infection. Kaplan-Meier infection-free survival curves and the log-rank test were used for group comparisons. Logistic regression was used to identify variables associated with KPC-Kp infection. Among 1310 patients included, 166 were colonized at the end of follow-up. Forty-seven out of 118 patients colonized at start of follow-up developed infection (39.8%) versus 31 out of 48 patients colonized during follow-up (64.6%; P = 0.006). Variables associated with KPC-Kp infection in the logistic regression analysis were: colonization detection during follow-up (OR, 2.74; 95% CI, 1.07 to 7.04; P = 0.03), Giannella risk score (OR, 1.51; 95% CI, 1.32 to 1.73; P < 0.001), high-risk ward (OR, 4.77; 95% CI, 1.61 to 14.10; P = 0.005) and urological manipulation after admission (OR, 3.69; 95% CI, 1.08 to 12.60; P = 0.04). In 25 out of 31 patients (80.6%) colonized during follow-up who developed KPC-Kp infection, infection appeared within 15 days after colonization. The risk of KPC-Kp infection was higher when colonization is recently acquired during hospitalization. In this prospective study, we concluded that the timing of colonization was a factor to assess when considering empirical treatment for suspected KPC-Kp infection and prophylaxis or infection control. IMPORTANCE In this study, it was confirmed that patients who became colonized during hospitalization had a higher risk of developing KPC-Kp infection than hospitalized patients who were already colonized at the start of follow-up. Besides, the risk of infection in the group of patients who became colonized during follow-up was greater in the first weeks immediately after colonization was confirmed. Our findings support the need for designing preventive strategies for patients at the highest risk of infection development, including those admitted in high-risk hospital wards and those undergoing urological procedures.

Methodology Establishment and Application of VITEK Mass Spectrometry to Detect Carbapenemase-Producing Klebsiella pneumoniae.

The ability of VITEK mass spectrometry (MS) in detection of bacterial resistance is currently under exploration and evaluation. In this study, we developed and validated a VITEK MS method to rapidly test carbapenemase-producing Klebsiella pneumoniae (CPKP). Solvents, antibiotic concentrations, crystal conditions and times, centrifugation speeds, and other factors were optimized to design a rapid sample pretreatment process for CPKP detection by VITEK MS. The related parameters of the mass spectrum were adjusted on the instrument to establish an CPKP detection mode. 133 clinically isolated strains of CPKP in the microbiology laboratory at the Shenzhen People's Hospital from 2004 to 2017 were selected for accuracy evaluation. The fresh suspected strains from the microbiology laboratory in 2020 were used to complete the clinical verification. Two antibiotics, meropenem (MEM) and imipenem (IPM), were used as substrates. These two substrates were incubated with suspected CPKP, and the results were obtained by VITEK MS detection. Using this method, different types of CPKP showed different detection results and all the CPKP strains producing KPC-2 and IMP-4 carbapenemase were detected by VITEK MS. Thus, VITEK MS can be used for rapid detection of CPKP, especially for some common types of CPKP. This method provides high accuracy and speed of detection. Combined with its cost advantages, it can be intensely valuable in clinical microbiology laboratories after the standard operating procedures are determined.

Epidemiology and in vitro activity of ceftazidime-avibactam, meropenem-vaborbactam, imipenem-relebactam, eravacycline, plazomicin, and comparators against Greek carbapenemase-producing Klebsiella pneumoniae isolates.

BACKGROUND: The increase in carbapenem-resistant Klebsiella pneumoniae (CRKP) infections is of great concern because of limited treatment options. New antimicrobials were recently approved for clinical therapy. This study evaluated the epidemiology of carbapenemase-producing K. pneumoniae isolates collected at a Greek university hospital during 2017-2020, and their susceptibilities to ceftazidime-avibactam (CAZ/AVI), meropenem-vaborbactam (M/V), imipenem-relebactam (I/R), eravacycline, plazomicin, and comparators. METHODS: Minimum inhibitory concentrations (MICs) were evaluated by Etest. Only colistin MICs were determined by the broth microdilution method. Carbapenemase genes were detected by PCR. Selected isolates were typed by multilocus sequence typing (MLST). RESULTS: A total of 266 carbapenemase-producing K. pneumoniae strains were isolated during the 4-year study period. Among them, KPC was the most prevalent (75.6%), followed by NDM (11.7%), VIM (5.6%), and OXA-48 (4.1%). KPC-producing isolates belonged mainly to ST258 and NDM producers belonged to ST11, whereas OXA-48- and VIM producers were polyclonal. Susceptibility to tigecycline, fosfomycin, and colistin was 80.5%, 83.8%, and 65.8%, respectively. Of the novel agents tested, plazomicin was the most active inhibiting 94% of the isolates at ≤ 1.5 µg/ml. CAZ/AVI and M/V inhibited all KPC producers and I/R 98.5% of them. All OXA-48 producers were susceptible to CAZ/AVI and plazomicin. The novel ß-lactam/ß-lactamase inhibitors (BLBLIs) tested were inactive against MBL-positive isolates, while eravacycline inhibited 61.3% and 66.7% of the NDM and VIM producers, respectively. CONCLUSIONS: KPC remains the predominant carbapenemase among K. pneumoniae, followed by NDM. Novel BLBLIs, eravacycline, and plazomicin are promising agents for combating infections by carbapenemase-producing K. pneumoniae. However, the emergence of resistance to these agents highlights the need for continuous surveillance and application of enhanced antimicrobial stewardship.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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). CONCLUSION: 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.

Clinical and Microbiological Characterization of Carbapenem-Resistant Enterobacteriales: A Prospective Cohort Study.

Aim: We aim to depict the clinicoepidemiological and molecular information of carbapenem-resistant Enterobacteriales (CRE) in Chongqing, China. Methods: We performed a prospective, observational cohort study, recruiting inpatients diagnosed with CRE infections from June 1, 2018, to December 31, 2019. We carried out strain identification and molecular characterization of CRE. eBURST analysis was conducted to assess the relationships among the different isolates on the basis of their sequence types (STs) and associated epidemiological data using PHYLOViZ. Clinical parameters were compared between the carbapenemase-producing Enterobacteriales (CPE) and non-CPE group. Findings: 128 unique CRE isolates from 128 patients were collected during the study period: 69 (53.9%) CPE and 59 (46.1%) non-CPE. The majority of CPE isolates were bla KPC-2 (56.5%), followed by bla NDM (39.1%) and bla IMP (5.8%). Klebsiella pneumoniae carbapenemase (KPC)-producing clonal group 11 Klebsiella pneumoniae (K. pneumoniae) was the most common CPE. Antibiotic resistance was more frequent in the CPE group than in the non-CPE group. Independent predictors for CPE infection were ICU admission and hepatobiliary system diseases. Although, there was no significant difference in desirability of outcome ranking (DOOR) outcomes between the two groups. At 30 days after index culture, 35 (27.3% ) of these patients had died. Conclusion: CRE infections were related to high mortality and poor outcomes, regardless of CRE subgroups. CPE were associated with prolonged ICU stays and had different clinical and microbiological characteristics than non-CPE. The identification of CPE/non-CPE and CRE resistance mechanisms is essential for better guidance of the clinical administration of patients with CRE infections.

Combinatorial Antimicrobial Efficacy and Mechanism of Linalool Against Clinically Relevant Klebsiella pneumoniae.

Antibiotic-adjuvant combinatory therapy serves as a viable treatment option in addressing antibiotic resistance in the clinical setting. This study was carried out to assess and characterize the adjuvant potential and mode of action of linalool against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). Linalool exhibited bactericidal activity alone (11,250 µg/ml) and in combination with meropenem (5,625 µg/ml). Comparative proteomic analysis showed significant reduction in the number of cytoplasmic and membrane proteins, indicating membrane damage in linalool-treated KPC-KP cells. Upregulation of oxidative stress regulator proteins and downregulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that linalool increases the bacterial surface charge as well as the membrane permeability. Intracellular leakage of nucleic acid and proteins was detected upon linalool treatment. Scanning and transmission electron microscopies further revealed the breakage of bacterial membrane and loss of intracellular materials. Linalool induced oxidative stress by generating reactive oxygen species (ROS) which initiates lipid peroxidation, leading to damage of the bacterial membrane. This leads to intracellular leakage, eventually killing the KPC-KP cells. Our study demonstrated that linalool possesses great potential in future clinical applications as an adjuvant along with existing antibiotics attributed to their ability in disrupting the bacterial membrane by inducing oxidative stress. This facilitates the uptake of antibiotics into the bacterial cells, enhancing bacterial killing.

Efficacy of Fosfomycin and Its Combination With Aminoglycosides in an Experimental Sepsis Model by Carbapenemase-Producing Klebsiella pneumoniae Clinical Strains.

Carbapenemase-producing Klebsiella pneumoniae infections are an increasing global threat with scarce and uncertain treatment options. In this context, combination therapies are often used for these infections. The bactericidal and synergistic activity of fosfomycin plus amikacin and gentamicin was studied trough time-kill assays against four clonally unrelated clinical isolates of carbapenemase-producing K. pneumoniae, VIM-1, VIM-1 plus DHA-1, OXA-48 plus CTXM-15, and KPC-3, respectively. The efficacy of antimicrobials that showed synergistic activity in vitro against all the carbapenemase-producing K. pneumoniae were tested in monotherapy and in combination, in a murine peritoneal sepsis model. In vitro, fosfomycin plus amikacin showed synergistic and bactericidal effect against strains producing VIM-1, VIM-1 plus DHA-1, and OXA-48 plus CTX-M-15. Fosfomycin plus gentamicin had in vitro synergistic activity against the strain producing KPC-3. In vivo, fosfomycin and amikacin and its combination reduced the spleen bacterial concentration compared with controls groups in animals infected by K. pneumoniae producing VIM-1 and OXA-48 plus CTX-M-15. Moreover, amikacin alone and its combination with fosfomycin reduced the bacteremia rate against the VIM-1 producer strain. Contrary to the in vitro results, no in vivo efficacy was found with fosfomycin plus amikacin against the VIM-1 plus DHA-1 producer strain. Finally, fosfomycin plus gentamicin reduced the bacterial concentration in spleen against the KPC-3 producer strain. In conclusion, our results suggest that fosfomycin plus aminoglycosides has a dissimilar efficacy in the treatment of this severe experimental infection, when caused by different carbapenemase-producing K. pneumoniae strains. Fosfomycin plus amikacin or plus gentamycin may be useful to treat infections by OXA-48 plus CTX-M-15 or KPC-3 producer strains, respectively.

Diseminación interhospitalaria de Klebsiella pneumoniae ST512 productora de KPC-3. Detección por MALDI-TOF / Interhospital dissemination of KPC-3 producing-Klebsiella pneumoniae ST512. Detection by MALDI-TOF

Introducción/Objetivo: Describir un brote por Klebsiella pneumoniae (KPN) productora de KPC-3 y determinar la eficacia diagnóstica de MALDI-TOF en su detección. Métodos: Estudio retrospectivo de las KPN-KPC-3 aisladas en 2 hospitales de Ciudad Real. Se buscó el pico a 11,109kDa±15 en el espectro proporcionado por MALDI-TOF para KPN. Resultados: Se aislaron 156 cepas de KPN que portaban el gen blaKPC-3, con un único perfil perteneciente al ST512 (31 cepas estudiadas). Hubo un 25% de infectados. Un 84% tuvieron origen nosocomial o relacionado con la asistencia sanitaria. El 93% tenía alguna enfermedad de base (31% de exitus en el primer mes). La detección del pico mostró una sensibilidad del 90% y una especificidad del 100%. Conclusiones: Detectamos la diseminación clonal de una cepa de KPN ST512 productora de KPC-3 en 3 hospitales de Ciudad Real. Además, evidenciamos la rentabilidad de MALDI-TOF en la detección precoz de KPN-KPC.(AU)

Introduction/Objective: To describe an outbreak of KPC-3-producing Klebsiella pneumoniae (KPN) and determine the diagnostic efficacy of MALDI-TOF in its detection. Methods: Retrospective study of the KPC-3-KPN isolated in 2 hospitals in Ciudad Real. The peak at 11,109kDa±15 was sought in the KPN spectra provided by MALDI-TOF. Results: We isolated 156 KPN strains that carried the blaKPC-3 gene, with a unique profile belonging to ST512 (31 strains studied). There was 25% of infected patients, 84% were nosocomial or related to health care and 93% had some underlying disease (31% of exitus in the first month). The detection of the peak showed 90% sensitivity and 100% specificity. Conclusions: We detected the clonal spread of a KPN ST512 strain producing KPC-3 in 3 hospitals in Ciudad Real. In addition, we show the profitability of MALDI-TOF in the early detection of KPC-KPN.