Carbapenem Resistance Phenotype Research Articles

Sensitivity and specificity of Nanopore sequencing for detecting carbapenem and 3rd-generation cephalosporin-resistant Enterobacteriaceae in urine samples: Real-time simulation with public antimicrobial resistance gene database

ObjectivesTo evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing. MethodsDNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST). ResultsWe processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (<1 ng/μl) for a final case of 53 in total. The sensitivity of antimicrobial resistance gene detection within 6 h, the optimal duration from real-time simulation, of Nanopore sequencing for the diagnosis of carbapenem-resistant and ceftriaxone-resistant phenotypes was 73.9 % (95%CI 56.0–91.9 %) and 81.1 % (95%CI 68.5–93.7 %), while the specificity was 96.7 % (95%CI 90.2–100.0 %) and 56.3 % (95%CI 31.9–80.6 %), respectively. The median times for MinION to generate DNA reads containing carbapenemase and ESBL/AmpC genes were 93 min (IQR 17–245.5) and 99 min (IQR 31.25–269.75) after sequencing commencement, respectively. ConclusionsNanopore sequencing can identify bacterial genotypic resistance in urine and may enable clinicians to adjust antimicrobial therapy earlier than routine AST.

Incidence, antimicrobial resistance and mortality of Klebsiella pneumoniae bacteraemia in Shanghai, China, 2018–2022

Background Klebsiella pneumoniae (KP) accounts for high antimicrobial resistance and mortality rates of bloodstream infections (BSIs). Objectives To investigate incidence, antimicrobial resistance and risk factors for mortality of KP BSIs in East China. Methods A retrospective study of patients with KP BSIs was conducted in a tertiary care hospital from 2018 to 2022. Medical records of all hospitalised patients with KP BSIs were reviewed and analysed. The incidence, antimicrobial resistance and mortality of KP BSIs were evaluated. The Kaplan-Meier method was used to plot survival curves and logistic regression was used to analyse risk factors for crude 30-day mortality. Results A total of 379 inpatients with KP BSIs were enrolled. The incidence of patients with KP BSIs was fluctuating between 4.77 and 9.40 per 100,000 patient-days. The crude 30-day mortality rate of these patients was 26.39%. Of the 379 KPisolates, 197 (51.98%) were carbapenem-resistant (CR) and 252 (66.49%) were multidrug-resistant (MDR). All isolates showed the lowest resistance to tigecycline (13.77%) and polymyxin B (14.61%). Cases with MDR/CR isolates had significantly longer length of hospital stay, higher crude 30-day mortality and medical costs than non-MDR/non-CR isolates. Age, CR phenotype, paracentesis, indwelling central venous catheter (CVC), use of carbapenems, tetracyclines, polymyxins B, and irrational empiric treatment were independently associated with crude 30-day mortality. Conclusion MDR/CR KP BSIs are associated with increased mortality, healthcare costs and prolonged hospitalisation. Patients with advanced age, CR phenotype, paracentesis, CVC, exposure to some antibiotics, and irrational empirical antibiotic treatment are at higher mortality risk.

Comparative In Vitro Activity of Ceftazidime-Avibactam, Imipenem-Relebactam, and Meropenem-Vaborbactam against Carbapenem-Resistant Clinical Isolates of Klebsiella pneumoniae and Pseudomonas aeruginosa.

Co-infection with carbapenem-resistant Klebsiella pneumoniae (CRKP) and Pseudomonas aeruginosa (CRPA) is associated with poor outcomes and historically relied on combination therapy with toxic agents for management. However, several novel β-lactam/β-lactamase inhibitor combination agents have been developed, offering potential monotherapy options. Here, we compare the in vitro activity of ceftazidime-avibactam (CZA), imipenem-relebactam (IRL), and meropenem-vaborbactam (MVB) against both CRKP and CRPA clinical isolates. Minimum inhibitory concentrations (MICs) for each agent were determined using broth microdilution. Carbapenemase gene detection was performed for representative isolates of varying carbapenem resistance phenotypes. IRL demonstrated excellent activity against CRKP and CRPA with susceptibility rates at 95.8% and 91.7%, respectively. While CZA and MVB showed comparable susceptibility to IRL against CRKP (93.8%), susceptibility of CRPA to CZA was modest at 79.2%, whereas most CRPA strains were resistant to MVB. Of the 35 CRKP isolates tested, 91.4% (32/35) carried a blaKPC gene. Only 1 of 37 (2.7%) CRPA isolates tested carried a blaVIM gene, which conferred phenotypic resistance to all three agents. None of the CRKP strains were cross-resistant to all three agents. Source of infection and co-infection did not significantly influence antimicrobial activity for IRL and CZA; none of the CRPA isolates from co-infected patients were susceptible to MVB. Our results suggest that novel β-lactam agents with antipseudomonal activity and stability against carbapenemases, such as IRL and CZA, offer potential monotherapy options for the treatment of co-infection involving both CRKP and CRPA, but not MVB.

Prevalence and characteristics of ertapenem-mono-resistant isolates among carbapenem-resistant Enterobacterales in China

ABSTRACT Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the optimal therapy options remain uncertain. To investigate the prevalence and characteristics of ETP-mono-resistant CRE, CRE strains were systematically collected from 102 hospitals across China between 2018 and 2021. A 1:1 randomized matching study was conducted with ETP-mono-resistant strains to meropenem- and/or imipenem-resistant (MEM/IPM-resistant) strains. Antimicrobial susceptibility testing, whole-genome sequencing, carbapenem-hydrolysing activity and the expression of carbapenemase genes were determined. In total, 18.8% of CRE strains were ETP-mono-resistant, with relatively low ertapenem MIC values. ETP-mono-resistant strains exhibited enhanced susceptibility to β-lactams, β-lactam/β-lactamase inhibitor combinations, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains (P < 0.05). Phylogenetic analysis revealed high genetic diversity among ETP-mono-resistant strains. Extended-spectrum β-lactamases (ESBLs) and/or AmpC, as well as porin mutations, were identified as potential major mechanisms mediating ETP-mono-resistance, while the presence of carbapenemases was found to be the key factor distinguishing the carbapenem-resistant phenotypes between the two groups (P < 0.001). Compared with the MEM/IPM-resistant group, limited carbapenemase-producing CRE (CP-CRE) strains in the ETP-mono-resistant group showed a significantly lower prevalence of ESBLs and porin mutations, along with reduced expression of carbapenemase. Remarkably, spot assays combined with modified carbapenem inactivation method indicated that ETP-mono-resistant CP-CRE isolates grew at meropenem concentrations eightfold above their corresponding MIC values, accompanied by rapidly enhanced carbapenem-hydrolysing ability. These findings illustrate that ETP-mono-resistant CRE strains are relatively prevalent and that caution should be exercised when using meropenem alone for treatment. The detection of carbapenemase should be prioritized.

Carbapenem-resistant Acinetobacter baumannii carrier detection: a simple and efficient protocol.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a substantial cause of nosocomial infections, classified among the most significant multidrug-resistant pathogens by the World Health Organization and by the US Centers for Disease Control. Limiting the spread of CRAB is an important goal of infection control, but laboratory methods for identification of CRAB carriers are not standardized. In this work, we compared different selective agar plates, tested the efficiency of A. baumannii identification by PCR for species-specific genes, and used PCR-based detection of common resistance genes to confirm the carbapenem-resistant phenotype. During a prospective study, we also determined the optimal sample enrichment time. Based on our results, we propose a simple and efficient protocol for the detection of CRAB carriage using skin sampling, short enrichment, selection on appropriate agar plates, and PCR-based identification, resulting in a turn-around time of 24 hours.

Characterization of an ST38 carbapenem-resistant and highly virulent Escherichia coli carrying conjugatively transferable ColV virulence-resistance and blaNDM-5-positive resistance plasmids.

To characterize an Escherichia coli strain causing bloodstream infection encoding both high-virulence and carbapenem-resistance phenotypes. Antimicrobial susceptibility testing, WGS and bioinformatics analysis were performed to characterize strain E1. The function of the ColV plasmid was investigated by the Galleria mellonella infection model, serum killing and macrophage killing assays. The fitness effect of the ColV plasmid was tested by growth curve, plasmid stability tests and the in vitro competition assay. The conjugation assay was performed to test the transferability of the ColV and blaNDM-5-carrying plasmids. E. coli E1 from bloodstream infection was MDR and highly virulent in the G. mellonella infection model. It belonged to phylogroup D, ST38 and serotype O7:H8. E1 carried a conjugatively transferable IncI1-type blaNDM-5-positive plasmid, which conferred carbapenem resistance, a conjugative IncFIB/FII-type ColV plasmid encoding an array of virulence-associated genes and antibiotic resistance genes blaTEM-1B, strAB and sul2, and seven other plasmids. Co-transfer of the ColV plasmid and the blaNDM-5-positive plasmid was observed. The ColV virulence-resistance hybrid plasmid contributed to the virulence, resistance to serum killing, and macrophage phagocytosis in E. coli E1. The carriage of this ColV plasmid did not constitute an in vitro fitness burden to strain E1 but caused fitness costs to E. coli strain EC600. The emergence of such a highly virulent and resistant strain with conjugative blaNDM-5-positive and ColV plasmids posed a significant threat to public health. Implementation of control measures is needed to prevent such strains from further disseminating in hospital settings and the community.

Multilocus sequence typing analysis and molecular characterization of carbapenemase related genes in Acinetobacter baumannii isolated from hospitalized patients in Erbil city, Iraq.

Acinetobacter baumannii, has been recognized by (WHO) as a global priority pathogen. It has been demonstrated to quickly pick up antimicrobial resistance genes. Multilocus sequence typing (MLST) is an unambiguous typing method for identifying accurate and portable nucleotide sequences of internal fragments of multiple housekeeping genes. The present study aimed to determine the sequence type using MLST, genetically define the carbapenem resistance phenotype and clarify the epidemiology of multidrug resistant (MDR) A. baumannii in the Kurdistan region of Iraq. Clinical samples were collected from ICU patients. VITEK 2 compact system was used for bacterial identification and antimicrobial susceptibility profile. PCR was used for detecting carbapenemase-related genes. Additionally, MLST was used to evaluate the genetic diversity of carbapenem-resistant isolates using Oxford scheme primers. In this investigation, 63 non-duplicate A. baumannii isolates from hospitalized patients were identified. According to CLSI standards, 75% and 73.4% of isolates were resistant to meropenem and imipenem respectively. Tigecycline and colistin were the most effective antibacterial agents. Of the various combinations of carbapenemase genes identified, the most common co-existence of genes present among clinical isolates were BlaOXA-23, and BlaOXA-51 (95.31%). While the less common combination of genes was detected in 4 isolates (6.25 %) consisting of the co-existence of all tested carbapenemase genes in the present study (BlaOXA-23, BlaOXA-51, BlaOXA-58, BlaIMP, BlaVIM, BlaNDM). Regarding MLST analysis, the results confirmed that ST 556 (n=4) had the greatest frequency rate among clinical isolates, followed by ST 218 (n=3). Notably, the most common worldwide clonal lineage was CC92, which corresponded to (ICII). Only two isolates from ST 441, on the other hand, matched CC109/ ICI. The present investigation revealed a significant level of diversity among A. baumannii isolates.

Prevalence of the virulence genes and their correlation with carbapenem resistance amongst the Pseudomonas aeruginosa strains isolated from a tertiary hospital in China.

Pseudomonas aeruginosa is one of the top-listed pathogens in nosocomial infection. It is notorious for its complicated virulence system and rapid adaptability to drugs or antimicrobials. In this study, we aimed to evaluate the prevalence of sixteen virulence genes in four groups including type III secretion system, biofilm formation, extracellular toxin biosynthesis and enzymes amongst 209 clinical Pseudomonas aeruginosa strains. We investigated the different distribution patterns of virulence genotypes based on carbapenem-resistant phenotype or the carriage of carbapenemase genes. The detection rate of each virulence gene varied greatly. phzM and plcN were detected in all collected strains, while pilB and exoU were only carried by a small portion of isolates (6.7% and 16.3%). Additionally, the number of genotypes observed in each group of examined virulence genes ranged from 4 to 8. Only the distribution of genotypes of type III secretion system showed statistical difference between carbapenem-mediated or carbapenem-resistant and carbapenem-sensitive strains. The virulence genotype of Pseudomonas aeruginosa was possibly interrelated to its resistance mechanism. Further research suggested that one particular TTSS genotype exhibited higher ratio in carbapenemase-producing strains and exoS was less frequently detected in CRPA strains carrying carbapenemase gene. Generally, the significant genetic diversity of virulence genes amongst Pseudomonas aeruginosa strains was highlighted in this study. Specific TTSS genotypes were associated with carbapenem-resistance. In particular, certain incompatibility might exist between exoS and carbapenemase genes, which provided valuable information for further understanding the relationship between carbapenem resistance and virulence.

Direct prediction of carbapenem resistance in Pseudomonas aeruginosa by whole genome sequencing and metagenomic sequencing.

Carbapenem resistance is a major concern in the management of antibiotic-resistant Pseudomonas aeruginosa infections. The direct prediction of carbapenem-resistant phenotype from genotype in P. aeruginosa isolates and clinical samples would promote timely antibiotic therapy. The complex carbapenem resistance mechanism and the high prevalence of variant-driven carbapenem resistance in P. aeruginosa make it challenging to predict the carbapenem-resistant phenotype through the genotype. In this study, using whole genome sequencing (WGS) data of 1,622 P. aeruginosa isolates followed by machine learning, we screened 16 and 31 key gene features associated with imipenem (IPM) and meropenem (MEM) resistance in P. aeruginosa, including oprD(HIGH), and constructed the resistance prediction models. The areas under the curves of the IPM and MEM resistance prediction models were 0.906 and 0.925, respectively. For the direct prediction of carbapenem resistance in P. aeruginosa from clinical samples by the key gene features selected and prediction models constructed, 72 P. aeruginosa-positive sputum samples were collected and sequenced by metagenomic sequencing (MGS) based on next-generation sequencing (NGS) or Oxford Nanopore Technology (ONT). The prediction applicability of MGS based on NGS outperformed that of MGS based on ONT. In 72 P. aeruginosa-positive sputum samples, 65.0% (26/40) of IPM-insensitive and 63.2% (24/38) of MEM-insensitive P. aeruginosa were directly predicted by NGS-based MGS with positive predictive values of 0.897 and 0.889, respectively. By the direct detection of the key gene features associated with carbapenem resistance of P. aeruginosa, the carbapenem resistance of P. aeruginosa could be directly predicted from cultured isolates by WGS or from clinical samples by NGS-based MGS, which could assist the timely treatment and surveillance of carbapenem-resistant P. aeruginosa.

Multi-approach methods to predict cryptic carbapenem resistance mechanisms in Klebsiella pneumoniae detected in Central Italy.

The rapid emergence of carbapenem-resistant Klebsiella pneumoniae (Kp) strains in diverse environmental niches, even outside of the clinical setting, poses a challenge for the detection and the real-time monitoring of novel antimicrobial resistance trends using molecular and whole genome sequencing-based methods. The aim of our study was to understand cryptic resistance determinants responsible for the phenotypic carbapenem resistance observed in strains circulating in Italy by using a combined approach involving whole genome sequencing (WGS) and genome-wide association study (GWAS). In this study, we collected 303 Kp strains from inside and outside clinical settings between 2018-2022 in the Abruzzo region of Italy. The antimicrobial resistance profile of all isolates was assessed using both phenotypic and bioinformatic methods. We identified 11 strains resistant to carbapenems, which did not carry any known genetic determinants explaining their phenotype. The GWAS results showed that incongruent carbapenem-resistant phenotype was associated specifically with strains with two capsular types, KL13 and KL116 including genes involved in the capsule synthesis, encoding proteins involved in the assembly of the capsule biosynthesis apparatus, capsule-specific sugar synthesis, processing and export, polysaccharide pyruvyl transferase, and lipopolysaccharide biosynthesis protein. These preliminary results confirmed the potential of GWAS in identifying genetic variants present in KL13 and KL116 that could be associated with carbapenem resistance traits in Kp. The implementation of advanced methods, such as GWAS with increased antimicrobial resistance surveillance will potentially improve Kp infection treatment and patient outcomes.

Decreasing Antibiotic Resistance Trends Nationally in Gram-Negative Bacteria Across United States Veterans Affairs Medical Centers, 2011-2020.

Gram-negative resistance is a well-acknowledged public health threat. Surveillance data can be used to monitor resistance trends and identify strategies to mitigate their threat. The objective of this study was to assess antibiotic resistance trends in Gram-negative bacteria. The first cultures of Pseudomonas aeruginosa, Citrobacter, Escherichia coli, Enterobacter, Klebsiella, Morganella morganii, Proteus mirabilis, and Serratia marcescens per hospitalized patient per month collected from 125 Veterans Affairs Medical Centers (VAMCs) between 2011 to 2020 were included. Time trends of resistance phenotypes (carbapenem, fluoroquinolone, extended-spectrum cephalosporin, multi-drug, and difficult-to-treat) were analyzed with Joinpoint regression to estimate average annual percent changes (AAPC) with 95% confidence intervals and p values. A 2020 antibiogram of reported antibiotic percent susceptibilities was also created to evaluate resistance rates at the beginning of the COVID-19 pandemic. Among 40 antimicrobial resistance phenotype trends assessed in 494,593 Gram-negative isolates, there were no noted increases; significant decreases were observed in 87.5% (n = 35), including in all P. aeruginosa, Citrobacter, Klebsiella, M. morganii, and S. marcescens phenotypes (p < 0.05). The largest decreases were seen in carbapenem-resistant phenotypes of P. mirabilis, Klebsiella, and M. morganii (AAPCs: - 22.9%, - 20.7%, and - 20.6%, respectively). In 2020, percent susceptibility was over 80% for all organisms tested against aminoglycosides, cefepime, ertapenem, meropenem, ceftazidime-avibactam, ceftolozane-tazobactam, and meropenem-vaborbactam. We observed significant decreases in antibiotic resistance for P. aeruginosa and Enterobacteralesover the past decade. According to the 2020 antibiogram, in vitro antimicrobial activity was observed for most treatment options. These results may be related to the robust infection control and antimicrobial stewardship programs instituted nationally among VAMCs.

Evaluation of In Vitro Activity of Ceftolozane/Tazobactam and Ceftazidime/Avibactam Against Carbapenem-Resistant Pseudomonas aeruginosa Strains and Mechanisms of Carbapenem Resistance: Data from Tertiary Care Hospital

Background: Carbapenem-resistant Pseudomonas aeruginosa is an endemic problem in several countries, notably Turkey. Objectives: This study aimed to investigate the underlying mechanisms contributing to the carbapenem resistance phenotype and enhance the in-vitro activity of ceftolozane-tazobactam (C/T) and ceftazidime-avibactam (C/A) against carbapenem-resistant P. aeruginosa strains. Methods: A total of 114 carbapenem-resistant P. aeruginosa strains were isolated from different types of clinical specimens. The tested antibiotics were evaluated using the antibiotic disk diffusion method. Additionally, C/T and C/A were tested using the gradient test method. The efficacy of phenylalanine-arginine-β-naphthylamide (PAβN) as efflux pump inhibitors was assessed to determine their ability to reduce meropenem minimum inhibitory concentrations. Polymerase chain reaction (PCR) assays were conducted to identify blaIMP, blaVIM, and blaNDM-1. Results: Among 114 strains of carbapenem-resistant P. aeruginosa, overall resistance rates for C/T and C/A were 10.7% and 8.8%, respectively. Efflux pump inhibitor-based antibiotic susceptibility testing indicated that 35.08% of strains showed resistance modulated by PAβN. Among the strains, 27 (24.5%) were found to produce metallo-beta-lactamase (MBL), with blaVIM (17 strains, 14.91%) being the most common, followed by blaIMP (12 strains, 10.53%). Conclusions: Emerging carbapenem resistance in P. aeruginosa strains is a serious therapeutic challenge for clinicians. Carbapenem resistance can be influenced by various factors, some of which were not assessed in our study. Nonetheless, our results revealed that the main mechanism associated with carbapenem-resistant P. aeruginosa strains is a PAβN-sensitive efflux pump. Among acquired MBLs, VIM-type enzymes were found to be the most prevalent.

Interplay between OXA-10 β-Lactamase Production and Low Outer-Membrane Permeability in Carbapenem Resistance in Enterobacterales.

The OXA-10 class D β-lactamase has been reported to contribute to carbapenem resistance in non-fermenting Gram-negative bacilli; however, its contribution to carbapenem resistance in Enterobacterales is unknown. In this work, minimum inhibitory concentrations (MICs), whole genome sequencing (WGS), cloning experiments, kinetic assays, molecular modelling studies, and biochemical assays for carbapenemase detection were performed to determine the impact of OXA-10 production on carbapenem resistance in two XDR clinical isolates of Escherichia coli with the carbapenem resistance phenotype (ertapenem resistance). WGS identified the two clinical isolates as belonging to ST57 in close genomic proximity to each other. Additionally, the presence of the blaOXA-10 gene was identified in both isolates, as well as relevant mutations in the genes coding for the OmpC and OmpF porins. Cloning of blaOXA-10 in an E. coli HB4 (OmpC and OmpF-deficient) demonstrated the important contribution of OXA-10 to increased carbapenem MICs when associated with porin deficiency. Kinetic analysis showed that OXA-10 has low carbapenem-hydrolysing activity, but molecular models revealed interactions of this β-lactamase with the carbapenems. OXA-10 was not detected with biochemical tests used in clinical laboratories. In conclusion, the β-lactamase OXA-10 limits the activity of carbapenems in Enterobacterales when combined with low permeability and should be monitored in the future.

Epidemiological and Molecular Characteristics of bla NDM-1 and bla KPC-2 Co-Occurrence Carbapenem-Resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged and spread worldwide. It can usually cause a serious threat complicating treatment options in clinical settings. However, treatment options are limited. The present study investigates the prevalence and genetic characteristics ofbla NDM-1 and bla KPC-2co-harboring clinical isolates ofKlebsiella pneumoniae. In this study, Multiplex polymerase chain reaction (PCR) was performed to detect the carbapenem-resistant genes, and the broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of antibacterial drugs. The transferability of carbapenem-resistant phenotypes was examined using filter mating assays. Overall, we used Illumina sequencing to evaluate the epidemiological and molecular characteristics of bla NDM-1 and bla KPC-2 (genes encoding carbapenemase) co-occurrence in CRKP strains. All strains exhibited resistance to carbapenems and other antibiotics. However, they were still susceptible to polymyxin E. Among them, 18 isolates were positive for bla KPC-2, bla NDM-1, and multiple virulence determinants, such as genes encoding the virulence factor aerobactin, yersiniabactin, and the regulator of the mucoid phenotype (rmpA and rmpA2). Whole genome sequencing revealed that the 18 CRKP strains belonged to ST11 and capsular serotype KL64, and could be grouped into two evolutionary branches. Furthermore, these strains displayed hypervirulence potential since all of them carried pLVPK-like plasmid. These findings suggested that ST11-KL64 CRKP strains are major threats in terms of nosocomial infections in this hospital. Hence, new strategies should be urgently developed to monitor, diagnose, and treat this high-risk CRKP clone.

Rapid Prediction of Multidrug-Resistant Klebsiella pneumoniae through Deep Learning Analysis of SERS Spectra.

Klebsiella pneumoniae is listed by the WHO as a priority pathogen of extreme importance that can cause serious consequences in clinical settings. Due to its increasing multidrug resistance all over the world, K. pneumoniae has the potential to cause extremely difficult-to-treat infections. Therefore, rapid and accurate identification of multidrug-resistant K. pneumoniae in clinical diagnosis is important for its prevention and infection control. However, the limitations of conventional and molecular methods significantly hindered the timely diagnosis of the pathogen. As a label-free, noninvasive, and low-cost method, surface-enhanced Raman scattering (SERS) spectroscopy has been extensively studied for its application potentials in the diagnosis of microbial pathogens. In this study, we isolated and cultured 121 K. pneumoniae strains from clinical samples with different drug resistance profiles, which included polymyxin-resistant K. pneumoniae (PRKP; n = 21), carbapenem-resistant K. pneumoniae, (CRKP; n = 50), and carbapenem-sensitive K. pneumoniae (CSKP; n = 50). For each strain, a total of 64 SERS spectra were generated for the enhancement of data reproducibility, which were then computationally analyzed via the convolutional neural network (CNN). According to the results, the deep learning model CNN plus attention mechanism could achieve a prediction accuracy as high as 99.46%, with robustness score of 5-fold cross-validation at 98.87%. Taken together, our results confirmed the accuracy and robustness of SERS spectroscopy in the prediction of drug resistance of K. pneumoniae strains with the assistance of deep learning algorithms, which successfully discriminated and predicted PRKP, CRKP, and CSKP strains. IMPORTANCE This study focuses on the simultaneous discrimination and prediction of Klebsiella pneumoniae strains with carbapenem-sensitive, carbapenem-resistant, and polymyxin-resistant phenotypes. The implementation of CNN plus an attention mechanism makes the highest prediction accuracy at 99.46%, which confirms the diagnostic potential of the combination of SERS spectroscopy with the deep learning algorithm for antibacterial susceptibility testing in clinical settings.

Clinical characteristics and outcomes of carbapenem-resistant Enterobacterales bacteremia in pediatric patients.

Clinical data on carbapenem-resistant Enterobacterales (CRE) bacteremia in the pediatric population are limited. This study investigated the clinical characteristics and outcomes of pediatric CRE bacteremia. Clinical data on bacteremia caused by carbapenem-susceptible and carbapenem-resistant Enterobacterales, including Escherichia coli, Klebsiella spp., Enterobacter spp., Serratia marcescens, Proteus mirabilis, Citrobacter spp., and Morganella spp., in pediatric patients from a children's hospital in Taiwan were retrospectively retrieved and analyzed. From January 2013 to December 2021, 471 clinical isolates of Enterobacterales bacteremia were identified in 451 episodes from 379 pediatric patients. Among all the isolates, the predominant species were E. coli (199/471, 42.2%), Klebsiella spp. (168/471, 35.6%), and Enterobacter spp. (59/471, 12.5%), with carbapenem-resistance rates of 1.5%, 11.9%, and 25.0%, respectively. Overall, 40 (8.4%) showed a carbapenem resistance phenotype. Patients' all-cause mortality rate at 14 days was significantly higher in CRE bacteremia episodes than non-CRE ones (12.5% vs. 3.6%, p<0.05). The predicting factor of a CRE bacteremia episode was the causative agent of Enterobacter spp. (adjusted OR of 2.551, CI 1.073-6.066, p<0.05) and ESBL-producing phenotype (adjusted OR 14.268, CI 5.120-39.762, p<0.001). Bloodstream infections caused by CRE are associated with a higher mortality rate in the pediatric population. Attention must be paid to preventing and managing pediatric patients with CRE infections.

Intestinal Carriage of Carbapenemase-Producing Enterobacteriaceae Members in Immunocompromised Children During COVID-19 Pandemic

Background: Hospital-acquired infection with carbapenem-resistant Enterobacteriaceae (CRE) is a global concern. The administration of antibiotics among the infected and non-infected immunocompromised children with SARS-CoV-2 is associated with an increased risk of intestinal CRE colonization and bacteremia during hospitalization. Objectives: The present study aimed to detect the correlation between the intestinal colonization of carbapenemase encoding Enterobacteriaceae with SARS-CoV-2 infection and antibiotic prescription among immunocompromised children admitted to the oncology and Bone Marrow Transplantation (BMT) wards. Methods: Stool samples were collected from the immunocompromised children, and the members of Enterobacteriaceae were isolated using standard microbiological laboratory methods. Carbapenem resistance isolates were initially characterized by the disc diffusion method according to CLSI 2021 and further confirmed by the PCR assay. SARS-CoV-2 infection was also recorded according to documented real-time PCR results. Results: In this study, 102 Enterobacteriaceae isolates were collected from the stool samples. The isolates were from Escherichia spp. (59/102, 57.8%), Klebsiella spp. (34/102, 33.3%), Enterobacter spp. (5/102, 4.9%), Citrobacter spp. (2/102, 1.9%), and Serratia spp. (2/102, 1.9%). The carbapenem resistance phenotype was detected among 42.37%, 73.52%, 40%, 50%, and 100% of Escherichia spp., Klebsiella spp., Enterobacter spp., Citrobacter spp., and Serratia spp., respectively. Moreover, blaOXA-48 (49.1%) and blaNDM-1 (29.4%), as well as blaVIM (19.6%) and blaKPC (17.6%) were common in the CRE isolates. SARS-CoV-2 infection was detected in 50% of the participants; however, it was confirmed in 65.45% (36/55) of the intestinal CRE carriers. The administration of antibiotics, mainly broad-spectrum antibiotics, had a significant correlation with the CRE colonization in both the infected and non-infected children with SARS-CoV-2 infection. Conclusions: Regardless of the COVID-19 status, prolonged hospitalization and antibiotic prescription are major risk factors associated with the CRE intestinal colonization in immunocompromised children.

First identification of multidrug-resistant Acinetobacter bereziniae isolates harboring blaNDM-1 from hospitals in South China

This study is a first report on the identification of multidrug-resistant (MDR) Acinetobacter bereziniae among non-baumannii acinetobacters that had previously escaped automated laboratory detection, and characterize their clinical courses of infection at two tertiary-care hospitals in Shenzhen city, China (2015–2017). Herein, definitive identification by PCR was performed with universal and species-specific primers targeting 16S rDNA and rpoB genes, respectively, followed by Sanger sequencing and blast analysis. Antimicrobial susceptibility of A. bereziniae isolates was assessed accordingly. Three of the five identified A. bereziniae isolates exhibited carbapenem-resistance and were subjected to a multiplex PCR assay to detect drug-resistance genes. Sequences of the rpoB amplicon were aligned with curated sequences from global databases for phylogenetic analysis on evolutionary relations. Five clinical isolates of A. bereziniae were thereby re-identified, whose infections were primarily nosocomial. Automated identification and susceptibility testing systems (Phoenix-100 and VITEK 2) proved insufficient for discriminating A. bereziniae from other acinetobacters such as Acinetobacter baumannii and Acinetobacter guillouiae. Among these isolates, three exhibited carbapenem-resistant phenotypes indistinguishable from that of carbapenem-resistant A. baumannii. The carbapenem-resistant A. bereziniae isolates were subsequently confirmed to carry a blaNDM-1 (New Delhi metallo-β-lactamase-1) gene downstream of ISAba125. Phylogenetic analysis revealed that A. bereziniae isolates evolved slowly but independently in local habitats. A. bereziniae isolates are difficult to distinguish by traditional automated detection systems. PCR-based identification via amplification and sequencing of selected house-keeping genes provides sufficient resolution for discriminating the isolates.

In vitro antibacterial activities of cefiderocol against Gram-negative clinical strains isolated from China in 2020

Cefiderocol (CFDC) is a parenteral siderophore cephalosporin that is active against Gram-negative bacteria, including carbapenem-resistant isolates. We report the in vitro activity of CFDC and other antibiotics against 1738 clinical isolates of Gram-negative bacilli (GNB) provided by five medical centres in five provinces of China in 2020 METHODS: Antibiotic susceptibility testing was performed using the Clinical and Laboratory Standards Institute broth microdilution method. Against Pseudomonas aeruginosa and Acinetobacter Spp., the CFDC concentration inhibiting the growth of 90% of the isolates (MIC90) (0.5 μg/mL) was identical and did not change by the carbapenem resistance phenotype. The susceptibility rate of P. aeruginosa and Acinetobacter Spp. to CFDC was high (> 98%) and was similar against isolates with and without meropenem resistance. The MIC of CFDC for all Stenotrophomonas maltophilia isolates (20 isolates) was ≤1 μg/mL and the MIC90 was 0.12 μg/mL. Considerable differences were noted in the susceptibility to CFDC between all tested Enterobacterales isolates and meropenem-non-susceptible Enterobacterales isolates. The MIC90 of CFDC was 1 μg/mL for all tested Enterobacterales isolates and 8 μg/mL for meropenem-non-susceptible Enterobacterales isolates. CFDC demonstrated potent in vitro activity against a recent collection of clinical isolates, including meropenem-non-susceptible isolates, obtained from medical centres in mainland China.

Epidemiological characteristics and molecular evolution mechanisms of carbapenem-resistant hypervirulent Klebsiella pneumoniae.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), a type of Klebsiella pneumoniae (KP) that exhibits hypervirulence and carbapenem resistance phenotypes, can cause severe infections, both hospital- and community-acquired infections. CR-hvKP has brought great challenges to global public health and is associated with significant morbidity and mortality. There are many mechanisms responsible for the evolution of the hypervirulence and carbapenem resistance phenotypes, such as the horizontal transfer of the plasmid carrying the carbapenem resistance gene to hypervirulent Klebsiella pneumoniae (hvKP) or carbapenemase-producing Klebsiella pneumoniae (CRKP) acquiring a hypervirulence plasmid carrying a virulence-encoding gene. Notably, KP can evolve into CR-hvKP by acquiring a hybrid plasmid carrying both the carbapenem resistance and hypervirulence genes. In this review, we summarize the evolutionary mechanisms of resistance and plasmid-borne virulence as well as the prevalence of CR-hvKP.