Raman spectrum combined with deep learning for precise recognition of Carbapenem-resistant Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) is a major pathogen that poses a serious threat to human health. Unfortunately, currently, there are no effective measures to curb its rapid development. To address this, an in-depth study on the surface-enhanced Raman spectroscopy (SERS) of 22 strains of 7 categories of CRE using a gold silver composite SERS substrate was conducted. The residual networks with an attention mechanism to classify the SERS spectrum from three perspectives (pathogenic bacteria type, enzyme-producing subtype, and sensitive antibiotic type) were performed. The results show that the SERS spectrum measured by the composite SERS substrate was repeatable and consistent. The SERS spectrum of CRE showed varying degrees of species differences, and the strain difference in the SERS spectrum of CRE was closely related to the type of enzyme-producing subtype. The introduced attention mechanism improved the classification accuracy of the residual network (ResNet) model. The accuracy of CRE classification for different strains and enzyme-producing subtypes reached 94.0% and 96.13%, respectively. The accuracy of CRE classification by pathogen sensitive antibiotic combination reached 93.9%. This study is significant for guiding antibiotic use in CRE infection, as the sensitive antibiotic used in treatment can be predicted directly by measuring CRE spectra. Our study demonstrates the potential of combining SERS with deep learning algorithms to identify CRE without culture labels and classify its sensitive antibiotics. This approach provides a new idea for rapid and accurate clinical detection of CRE and has important significance for alleviating the rapid development of resistance to CRE.

Blood culture quality and turnaround time of clinical microbiology laboratories in Chinese Teaching Hospitals: A multicenter study.

PURPOSE: Blood culture (BC) remains the gold standard for the diagnosis of bloodstream infections. Improving the quality of clinical BC samples, optimizing BC performance, and accelerating antimicrobial susceptibility test (AST) results are essential for the early detection of bloodstream infections and specific treatments. METHODS: We conducted a retrospective multicenter study using 450,845 BC specimens from clinical laboratories obtained from 19 teaching hospitals between 1 January 2021 and 31 December 2021. We evaluated key performance indicators (KPIs), turnaround times (TATs), and frequency distributions of processing in BC specimens. We also evaluated the AST results of clinically significant isolates for four different laboratory workflow styles. RESULTS: Across the 10 common bacterial isolates (n = 16,865) and yeast isolates (n = 1011), the overall median (interquartile range) TATs of AST results were 2.67 (2.05-3.31) and 3.73 (2.98-4.64) days, respectively. The specimen collections mainly occurred between 06:00 and 24:00, and specimen reception and loadings mainly between 08:00 and 24:00. Based on the laboratory workflows of the BCs, 16 of the 19 hospitals were divided into four groups. Time to results (TTRs) from specimen collection to the AST reports were 2.35 (1.95-3.06), 2.61 (1.98-3.32), 2.99 (2.60-3.87), and 3.25 (2.80-3.98) days for groups I, II, III, and IV, respectively. CONCLUSION: This study shows the related BC KPIs and workflows in different Chinese hospitals, suggesting that laboratory workflow optimization can play important roles in shortening time to AST reports and initiation of appropriate timely treatment.

Sensitive and rapid detection of tet(X2) ~ tet(X5) by loop-mediated isothermal amplification based on visual OTG dye.

The emergence of tigecycline-resistant tet(X2/X3/X4/X5) genes poses a new threat to the efficacy of anti-infective therapy and the safety of our food and environment. To control the transfer of such genes, a sensitive and rapid molecular method is warranted to detect tet(X2/X3/X4/X5) genes in clinical isolates. Herein, we established a loop-mediated isothermal amplification (LAMP) assay to rapidly detect tet(X2/X3/X4/X5) genes, and the results were assessed by chromogenic visualization. The specificity and sensitivity of the primers during the LAMP assay for the simultaneous detection of tet(X2/X3/X4/X5) genes were determined in this study. All 48 clinical strains without tet(X2/X3/X4/X5) genes yielded negative results during the LAMP assay, substantiating the high specificity of the LAMP primers. The detection thresholds of this assay were 1.5 × 102 CFU/ml and 0.2 fg/uL corresponding to a 10 to 100-fold and 100-fold increase in sensitivity compared to polymerase chain reaction (PCR) assays. Out of 52 bacterial strains tested, using PCR as a reference, our research revealed that the LAMP assay demonstrated a sensitivity and specificity of 100%. To sum up, our novel approach has huge prospects for application in the simultaneous detection of tet(X2/X3/X4/X5) genes and can be applied to detect other drug-resistance genes.

Prognostic models for estimating severity of disease and predicting 30-day mortality of Hypervirulent Klebsiella pneumoniae infections: a bicentric retrospective study.

BACKGROUND: Hypervirulent Klebsiella pneumoniae (hvKP) is emerging globally and can cause various, severe infections in healthy individuals. However, the clinical manifestations of hvKP infections are nonspecific, and there is no gold standard for differentiating hvKP strains. Our objective was to develop prognostic models for estimating severity of disease and predicting 30-day all-cause mortality in patients with hvKP infections. METHODS: We enrolled 116 patients diagnosed with hvKP infections and obtained their demographic and clinical data. Taking septic shock and acute respiratory distress syndrome (ARDS) as the primary outcomes for disease severity and 30-day all-cause mortality as the primary outcome for clinical prognosis, we explored the influencing factors and constructed prognostic models. RESULTS: The results showed that increased Acute Physiologic and Chronic Health Evaluation (APACHE) II score [odds ratio (OR) = 1.146; 95% confidence interval (CI), 1.059-1.240], decreased albumin (ALB) level (OR = 0.867; 95% CI, 0.758-0.990), diabetes (OR = 9.591; 95% CI, 1.766-52.075) and high procalcitonin (PCT) level (OR = 1.051; 95%CI, 1.005-1.099) were independent risk factors for septic shock. And increased APACHE II score (OR = 1.254; 95% CI, 1.110-1.147), community-acquired pneumonia (CAP) (OR = 11.880; 95% CI, 2.524-55.923), and extrahepatic lesion involved (OR = 14.718; 95% CI, 1.005-215.502) were independent risk factors for ARDS. Prognostic models were constructed for disease severity with these independent risk factors, and the models were significantly correlated with continuous renal replacement therapy (CRRT) duration, vasopressor duration, mechanical ventilator duration and length of ICU stay. The 30-day all-cause mortality rate in our study was 28.4%. Younger age [hazard ratio (HR) = 0.947; 95% CI, 0.923-0.973)], increased APACHE II score (HR = 1.157; 95% CI, 1.110-1.207), and decreased ALB level (HR = 0.924; 95% CI, 0.869-0.983) were the independent risk factors for 30-day all-cause mortality. A prediction model for 30-day mortality was constructed, which had a good validation effect. CONCLUSIONS: We developed validated models containing routine clinical parameters for estimating disease severity and predicting 30-day mortality in patients with hvKP infections and confirmed their calibration. The models may assist clinicians in assessing disease severity and estimating the 30-day mortality early.

Rapid identification of the resistance of urinary tract pathogenic bacteria using deep learning-based spectroscopic analysis.

The resistance of urinary tract pathogenic bacteria to various antibiotics is increasing, which requires the rapid detection of infectious pathogens for accurate and timely antibiotic treatment. Here, we propose a rapid diagnosis strategy for the antibiotic resistance of bacteria in urinary tract infections (UTIs) based on surface-enhanced Raman scattering (SERS) using a positively charged gold nanoparticle planar solid SERS substrate. Then, an intelligent identification model for SERS spectra based on the deep learning technique is constructed to realize the rapid, ultrasensitive, and non-labeled detection of pathogenic bacteria. A total of 54,000 SERS spectra were collected from 18 isolates belonging to 6 species of common UTI bacteria in this work to realize identification of bacterial species, antibiotic sensitivity, and multidrug resistance (MDR) via convolutional neural networks (CNN). This method significantly simplify the Raman data processing processes without background removing and smoothing, however, achieving 96% above classification accuracy, which was significantly greater than the 85% accuracy of the traditional multivariate statistical analysis algorithm principal component analysis combined with the K-nearest neighbor (PCA-KNN). This work clearly elucidated the potential of combining SERS and deep learning technique to realize culture-free identification of pathogenic bacteria and their associated antibiotic sensitivity.

Epidemiology of Carbapenem-Resistant Enterobacteriaceae Infections: Report from the China CRE Network.

Carbapenem-resistant Enterobacteriaceae (CRE) infection is highly endemic in China, but estimates of the infection burden are lacking. We established the incidence of CRE infection from a multicenter study that covered 25 tertiary hospitals in 14 provinces. CRE cases defined as carbapenem-nonsusceptible Citrobacter freundii, Escherichia coli, Enterobacter cloacae, or Klebsiella pneumoniae infections during January to December 2015 were collected and reviewed from medical records. Antimicrobial susceptibility testing and carbapenemase gene identification were performed. Among 664 CRE cases, most were caused by K. pneumoniae (73.9%), followed by E. coli (16.6%) and E. cloacae (7.1%). The overall CRE infection incidence per 10,000 discharges was 4.0 and differed significantly by region, with the highest in Jiangsu (14.97) and the lowest in Qinghai (0.34). Underlying comorbidities were found in 83.8% of patients; the median patient age was 62 years (range, 45 to 74 years), and 450 (67.8%) patients were male. Lower respiratory tract infections (65.4%) were the most common, followed by urinary tract infection (16.6%), intra-abdominal infection (7.7%), and bacteremia (7.7%). The overall hospital mortality rate was 33.5%. All isolates showed nonsusceptibility to carbapenems and cephalosporins. The susceptibility rate of polymyxin B was >90%. Tigecycline demonstrated a higher susceptibility rate against E. coli than against K. pneumoniae (90.9% versus 40.2%). Of 155 clinical isolates analyzed, 89% produced carbapenemases, with a majority of isolates producing KPC (50%) or NDM (33.5%)-type beta-lactamases among K. pneumoniae and E. coli The incidence of CRE infection in China was 4.0 per 10,000 discharges. The patient-based disease burden in tertiary hospitals in China is severe, suggesting an urgent need to enhance infection control.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.

Genome-Based Analysis of a Multidrug-Resistant Hypervirulent Klebsiella pneumoniae.

Reports on multidrug-resistant hypervirulent Klebsiella pneumoniae (MDR-hvKP) in recent years indicate the wide-spreading trend of MDR-hvKP. The co-occurrence of hypervirulence and drug resistance poses a great challenge to clinical treatment. In this study, molecular characteristics of an MDR strain hvKP247 and 30 clinically isolated hvKP strains were characterized. The whole genome of hvKP247 belonging to sequence type (ST) 5214 and capsule serotype K1 was sequenced and analyzed. Conjugation experiments were performed to evaluate transferability of the plasmids in hvKP247. We found two new STs among our isolates, ST5570 and ST5571. The ST5214 hvKP247 contained two transferable plasmids: a hybrid virulence plasmid (pHvKP247-vir) carrying transfer-related modules that had self-transferable ability, and a drug-resistant plasmid (pHvKP247-MDR) that could be indirectly transferred with the help of pHvKP247-vir. The virulence-related genes were located on the pHvKP247-vir and chromosomal ICEKp1 mobile genetic element. In conclusion, the hybrid virulence plasmid and the drug-resistant plasmid are co-transferred, which emphasizes the importance of raising public awareness of the simultaneous spread of virulence and resistance genes of MDR-hvKP strains.

Occurrence of High Levels of Cefiderocol Resistance in Carbapenem-Resistant Escherichia coli before Its Approval in China: a Report from China CRE-Network.

Cefiderocol has been approved in the United States and Europe but not in China. We aim to evaluate carbapenem-resistant Enterobacterales (CRE) susceptibility to cefiderocol to provide baseline data and investigate the resistance mechanism. From 2018 to 2019, 1,158 CRE isolates were collected from 23 provinces and municipalities across China. The MICs of antimicrobials were determined via the agar dilution and broth microdilution methods. Whole-genome sequencing was performed for 26 cefiderocol-resistant Escherichia coli isolates to investigate the resistance mechanism. Clone transformations were used to explore the function of cirA, pbp3, and blaNDM-5 in resistance. Among the 21 antimicrobials tested, aztreonam-avibactam had the highest antibacterial activity (98.3%), followed by cefiderocol (97.3%) and colistin (95.3%). A total of 26 E. coli isolates harboring New Delhi metallo-beta-lactamase 5 (NDM-5) showed high levels of cefiderocol resistance, of which sequence type 167 (ST167) accounted for 76.9% (20/26). We found 4 amino-acid insertions (YRIN/YRIK) at position 333 of penicillin-binding protein 3 (PBP3) in the 26 E. coli isolates, and 22 isolates had a siderophore receptor cirA premature stop codon. After obtaining the wild-type cirA supplementation, the MIC of the transformants decreased by 8 to 16 times in two cefiderocol-resistant isolates. A cefiderocol-susceptible isolate harboring NDM-5 has an MIC increased from 1 µg/mL to 64 µg/mL after cirA deletion, and the MIC decreased from 64 µg/mL to 0.5 µg/mL after blaNDM-5 deletion. The MIC of the E. coli DH5α, from which the pbp3 mutant was obtained, increased from 0.064 µg/mL to 0.25 µg/mL. Cefiderocol showed activity against most CRE in China. The resistance of ST167 E. coli to cefiderocol is a combination of the premature stop codon of cirA, pbp3 mutation, and blaNDM-5 existence. IMPORTANCE Cefiderocol, a new siderophore cephalosporin, has been approved in the United States and Europe but not in China. At present, there are almost no antimicrobial susceptibility evaluation data on cefiderocol in China. We evaluated the in vitro susceptibility of 1,158 strains of carbapenem-resistant Enterobacterales to cefiderocol and other antibiotics. We found that a high proportion of Escherichia coli showed high-level resistance to cefiderocol. Whole-genome sequencing (WGS) and molecular cloning experiments confirmed that the synergistic effect of the cirA gene premature stop codon, blaNDM-5 existence, and the pbp3 mutation is associated with high levels of cefiderocol resistance.

Microbiological Characteristics of Carbapenem-Resistant Enterobacteriaceae Clinical Isolates Collected from County Hospitals.

OBJECTIVE: To investigate the molecular characteristics of carbapenem-resistant Enterobacteriaceae (CRE) from county hospitals in China. MATERIALS AND METHODS: Forty-three sequential non-duplicate CRE strains (including 33 Klebsiella pneumoniae isolates, 4 Enterobacter cloacae isolates, 3 Escherichia coli isolates, 1 Serratia marcescens, 1 Morganella morganii and 1 Citrobacter freundii) were collected from 4 county hospitals and 2 municipal hospitals. Antimicrobial susceptibility testing was conducted by broth microdilution method, using 3-aminophenylboronic acid and EDTA and the modified carbapenem inactivation method (mCIM) to screen phenotype of carbapenemase. ß-Lactamases were characterized by polymerase chain reaction (PCR) and DNA sequencing. The transferability of bla NDM-5 was investigated by transformation experiment. Clonal relatedness was evaluated by pulsed-field gel electrophoresis and multilocus sequence typing . RESULTS: The results of antimicrobial susceptibility testing indicated that 43 CRE strains were resistant to most of the antimicrobial agents, except tigecycline and colistin. Overall, 93%, 93%, and 97.7% of these strains were resistant to imipenem, meropenem, and ertapenem, respectively. PCR and DNA sequencing indicated that 67.4% (29/43) were bla KPC-2 positive isolates, in which 3.4% (1/29) was coproduced with bla NDM-1. In addition, 7.0% (3/43), 4.7% (2/43), 4.7% (2/43), 2.3% (1/43), 2.3% (1/43) were bla NDM-1, bla NDM-16, bla NDM-4, bla NDM-5, bla IMP-4 positive isolates, respectively. The 29 bla KPC-2-positive isolates belonged to 12 different PFGE type and designated as ST11 (n=20) and ST15, ST39, ST116, ST667, ST2245, ST2338. The plasmid bearing bla NDM-5 could be transferred into recipient E. coli J53 through transformation. CONCLUSION: Our study indicated the dissemination of CRE between the tertiary hospitals and secondary hospitals.

The performance of galactomannan in combination with 1,3-ß-D-glucan or aspergillus-lateral flow device for the diagnosis of invasive aspergillosis: Evidences from 13 studies.

Galactomannan (GM), 1,3-ß-D-glucan (BDG) and aspergillus-lateral flow device (LFD) are recognized as diagnostic tools for invasive aspergillosis (IA). The combined performance of these assays, however, is inconsistent in various studies. We undertook a meta-analysis of 13 studies involving 1513 patients to evaluate the utility of GM in combination with BDG or LFD for diagnosing IA. The pooled SEN, SPE, PLR, NLR and diagnostic odds ratio (DOR) were calculated and constructed to summarize the overall combined performance. Combining both positive results of GM and BDG assays leaded to the pooled SEN 0.49 (95%CI 0.27-0.72), SPE 0.98 (95%CI 0.94-1.00), PLR 31.68 (95%CI 5.36-187.37), NLR 0.52 (95%CI 0.32-0.84) and DOR 61.23 (95%CI 6.96-538.90). Comparing with GM and BDG assays, both positive results of GM and LFD leaded to high SEN, similar SPE, low PLR and NLR. At least one positive result of GM or LFD conferred great SEN 0.93 and low NLR 0.08. Both positive results of GM and BDG or LFD assay were in favor of confirming the existence of IA. And both negative results of GM and LFD were beneficial to rule out IA. Further studies with sufficient sample size should focus on the diagnostic performance and cost-effectiveness of these combined tests in clinical setting.

Colonization prevalence and antibiotic susceptibility of Group B Streptococcus in pregnant women over a 6-year period in Dongguan, China.

This study investigated the prevalence of recto-vaginal Group B Streptococcus (GBS) colonization, serotype distribution, and antimicrobial susceptibility patterns among pregnant women in Dongguan, China. Recto-vaginal swabs were collected from pregnant women at gestational age 35-37 weeks between January 1st 2009 and December 31st 2014. Isolates were serotyped by latex-agglutination and were tested against seven antimicrobials by disk diffusion. Of 7,726 pregnant women who completed GBS testing, 636 (8.2%) were GBS carriers. Of 153 GBS isolates available for typing, 6 serotypes (Ia, Ib, III, V, VI and VIII) were identified with type III being predominant, while 9 (5.9%) were non-typable isolates. All isolates were sensitive to penicillin, ceftriaxone, linezolid and vancomycin, whereas 52.4% were resistant to clindamycin, 25.9% were resistant to levofloxacin and 64.9% were resistant to erythromycin. This study showed the recto-vaginal colonization prevalence of GBS in Dongguan is significant. Due to 100% susceptibility to penicillin of all GBS samples, penicillin remains the first recommendation for treatment and prevention against GBS infection. Susceptibility testing should be performed for women allergic to penicillin in order to choose the most appropriate antibacterial agents for treatment and prevention of vertical transmission to neonates. In addition, we suggest establishing standard processes for GBS culture and identification in China as early as possible.