This study aimed to investigate the molecular basis of polymyxin B resistance in clinical carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates. A total of 52 polymyxin B-resistant CRKP clinical isolates were collected at West China Hospital between 2018 and 2021. Polymyxin B susceptibility testing was performed using the broth microdilution method. Whole-genome sequencing was conducted to determine sequence types (STs), capsular loci, resistance genes, virulence genes and mutations in genes associated with polymyxin B resistance. Transcriptome sequencing was used to compare changes in gene expression between selected high-minimum inhibitory concentration (MIC) isolates after polymyxin B exposure and controls cultured without polymyxin B. The majority (98.08

Antibiotic tolerance enables bacteria to survive bactericidal antibiotic exposure and has been linked to resistance evolution in laboratory systems and individual infections, but its role in plasmid-mediated resistance evolution in clinical populations remains unclear. Here, we analyzed a longitudinal collection of more than 800 clinical Klebsiella pneumoniae isolates spanning 1997-2020. Among 779 minimum inhibitory concentration (MIC)-defined ertapenem-susceptible isolates, 137 (17.6%) displayed hidden ertapenem tolerance, defined by enhanced survival after 6 h at 30 times the isolate-specific ertapenem MIC, mostly without extended lag time or reduced growth rate. Tolerance was detected before local ertapenem introduction and was enriched among ertapenem-resistant isolates, supporting a population-level association between pre-existing tolerance and the emergence of carbapenem resistance. Genomic and plasmid-curing analyses separated plasmid-mediated carbapenem resistance from plasmid-independent antibiotic tolerance. Moreover, tolerant recipient backgrounds enhanced resistance plasmid acquisition, preserved viable recipients following antibiotic exposure and accelerated ceftazidime-avibactam resistance evolution. A phylogeny-guided variant-enrichment analysis further identified the uhpABC regulatory operon as a candidate tolerance-associated locus, and coordinated expression of the complete operon increased ertapenem survival. Together, these findings identify clinical antibiotic tolerance as a pre-existing, MIC-hidden phenotype that can facilitate plasmid-mediated carbapenem resistance evolution in K. pneumoniae.

Objective Mucormycosis is a life-threatening invasive fungal infection with high mortality,yet traditional diagnostic methods are limited by low positivity rates.This study aims to evaluate the diagnostic performance and clinical utility of metagenomic next-generation sequencing(mNGS)in mucormycosis.Methods A retrospective analysis was conducted on 135 patients with mNGS results positive for Mucorales fungi at West China Hospital of Sichuan University from November 1,2022,to October 31,2024.Based on comprehensive clinical diagnostic criteria(including proven and probable cases),patients were classified into a confirmed mucormycosis group and a non-mucormycosis group.Receiver operating characteristic(ROC)curve analysis was used to evaluate the diagnostic performance of normalized read counts(lgRPM)from different specimen types.Fungal species distribution and laboratory parameters were compared between the two groups.Results Among the 135 patients with positive mNGS results for Mucorales,100(74.1%)were ultimately diagnosed with mucormycosis.ROC curve analysis revealed that the diagnostic performance of mNGS varied by specimen type.For blood specimens,the area under the curve(AUC)was 0.772,with a specificity of 87.5%at the optimal cutoff value of 0.11 RPM.For bronchoalveolar lavage fluid specimens,the AUC was 0.717,with a sensitivity of 76.5%at the optimal cutoff value of 0.02 RPM.Combined analysis of all specimens showed that at the optimal cutoff value of 0.08 RPM(approximately 8 reads/100M),the sensitivity and specificity were 62.0%and 71.4%,respectively.Species distribution analysis showed that the proportions of Cunninghamella elegans(11.0%vs.2.9%)and Rhizomucor pusillus(9.0%vs.2.9%)were significantly higher in the confirmed group than in the non-mucormycosis group(P<0.05).Levels of C-reactive protein and interleukin-6 were also significantly higher in the confirmed group(P<0.05).Notably,all seven renal perfusion fluid samples yielded false-positive mNGS results.Conclusion mNGS technology can effectively improve the diagnostic yield for mucormycosis.However,results should be interpreted in conjunction with specimen type,read count,and clinical characteristics.BALF specimens offer high sensitivity,making them suitable for screening,while blood specimens demonstrate high specificity,making them valuable for confirmation.Positive results from low-biomass samples such as renal perfusion fluid warrant caution against false positivity.Fungal species identification and inflammatory markers may serve as adjunctive evidence for clinical diagnosis.

A 45-year-old male with a history of lumbar tuberculosis presented with a ruptured mycotic iliac artery aneurysm as the initial manifestation. Imaging demonstrated aneurysm rupture with pseudoaneurysm formation and concurrent disseminated lesions involving the vertebrae and soft tissues. Intraoperative specimens grew an Aspergillus fumigatus strain that exhibited highly atypical morphology: the colonies were albino‑like, slow‑growing, and non‑sporulating, differing markedly from the classic A. fumigatus phenotype. Peripheral blood metagenomic sequencing detected A. fumigatus, and the serum galactomannan antigen was markedly elevated. Molecular sequencing confirmed the isolate as A. fumigatus sequence type ST26 and identified the multidrug resistance‑associated gene ABCA. The final diagnosis was disseminated aspergillosis presenting as a ruptured mycotic iliac artery aneurysm, complicated by prosthetic graft infection and multiorgan dissemination. The patient received systemic antifungal therapy with voriconazole, along with adequate surgical drainage and debridement. Subsequently, his inflammatory markers declined gradually, and he was discharged on hospital day 58.

This study investigated the clinical and molecular characteristics of Staphylococcus aureus (S. aureus) isolates from patients with coronavirus disease 19 (COVID- 19) at West China Hospital between December 1, 2022 and January 31, 2023. In total, 102 strains isolated from sputum, bronchoalveolar lavage fluid, endotracheal aspirates, and blood were collected from 102 patients and subjected to multilocus sequence typing and antimicrobial susceptibility testing. Eighteen virulence genes were also analyzed by polymerase chain reaction. Seventy-five patients were discharged and 27 died. The predominant comorbidities were hypertension, diabetes mellitus, and cardiac disease. Twenty-eight known sequence types (STs) and 10 novel ones (ST8773/CC398, ST9221/CC5, ST9222/CC59, ST9223/CC8, ST9224/CC22, ST9225/CC1, ST9226/CC5, ST9227/CC59, ST9228/CC59, ST9229/CC398) were identified. The dominant molecular types were ST15 (CC15), ST59 (CC59), and ST5 (CC5). Among the three most prevalent STs, ST5 was significantly more resistant to levofloxacin, moxifloxacin, ciprofloxacin, and sulfamethoxazole than were ST59 and ST15. ST59 and ST5 had higher rates of resistance to erythromycin and clindamycin than ST15. All isolates contained at least eight virulence genes. The hemolysin gene hlb was found to be more prevalent in ST59 (100

Objective: Ceftazidime-avibactam (CZA) is a novel beta-lactam/ beta-lactamase inhibitor with activity against carbapenem-resistant Klebsiella pneumoniae (CRKP) that produce Klebsiella pneumoniae carbapenemase (KPC). In this study, we report the first cases of CZA resistance to develop during treatment of CRKP infections and identify the resistance mechanism. Methods: APB/EDTA and NG-Test CARBA5 were used to detect the production of carbapenemase, whole-genome sequencing (WGS) and conjugation experiment were used to identify potential resistance mechanisms of CZA-susceptible (HX1032) and- resistant (HX1192) K. pneumoniae isolates. Results: HX1192 K. pneumoniae was not recognized by APB/EDTA and NG-Test CARBA5 phenotypic assays, WGS revealed it carrying a novel KPC variant, KPC-179, molecular analysis highlighted a G394A mutation, and an ATC insertion at 543 in the bla KPC-2 gene, resulting in an A133T substitution and insertion of the amino acid S at Ambler position 183 in the protein sequence. Remarkably, this mutation restored susceptibility of imipenem (MIC = 0.25 mg/L). Conclusion: Our study highlights the importance of monitoring susceptibility during CZA treatment and accurately detecting KPC variants.

Background: Currently, EUCAST have issued guidelines for direct rapid antimicrobial susceptibility testing (RAST) on blood cultures. There were few reports on total laboratory automation (TLA) for RAST in China. Additionally, certain antibiotic discs used in China with specific concentrations lack EUCAST breakpoints. The purpose of this study is to use TLA to evaluate EUCAST RAST methods in Enterobacterales-containing blood cultures and to investigate the optimal breakpoints for a selection of antibiotics, including cefepime (EFP), cefotaxime (CTX), ceftazidime (CAZ), piperacillin-tazobactam (TZP), and cefoperazone/sulbactam (CSL). Methods: From April to August 2022, blood cultures positive for Enterobacterales (54 Escherichia coli and 60 Klebsiella pneumoniae) were analyzed. EUCAST RAST (4h, 6h, 8h) were performed using TLA and compared with Vitek 2 results. Results: EUCAST RAST's readable inhibition zone increased over time, with high categorical agreement with Vitek 2 (97.1%, 96.2%, 96.1% for E. coli , and 96.1%, 97.1%, 97.9% for K. pneumoniae) and low error rates. Based on the optimal breakpoints we defined, the categorical agreement for EFP, CTX, CAZ, and TZP against E. coli was greater than 90% at both 6 and 8 hours. However, the categorical agreement for CSL was 57.1% at 6 hours and 74.3% at 8 hours, primarily due to a higher proportion of minor errors (42.9% and 25.7%, respectively). For K. pneumoniae, the categorical agreement for all five antibiotics was greater than 90% at both time points. Conclusion: According to the CLSI-M52 standards, the detection performance of EUCAST RAST at 4h, 6h, and 8h was equivalent to that of Vitek 2. Except for ciprofloxacin in E. coli, the readable rates and categorical agreement of all antibiotics are good at 6h. For the optimal breakpoints we have established, all antibiotics except for CSL against E. coli achieve a categorical agreement of over 90%.

Objective:To investigate the clinical characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from patients with bloodstream infections in a large tertiary-care general hospital in Southwest China.Methods:A total of 131 strains of non-repeating CRKP were collected from the blood cultures of patients who had bloodstream infections in 2015-2019. The strains were identified by VITEK-2, a fully automated microbial analyzer, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The minimum inhibitory concentration (MIC) was determined by microbroth dilution method. The common carbapenemase resistant genes and virulence factors were identified by PCR. Homology analysis was performed by multilocus sequencing typing. Whole genome sequencing was performed to analyze the genomic characteristics of CRKP without carbapenemase.Results:The 131 strains of CRKP showed resistance to common antibiotics, except for polymyxin B (1.6% resistance rate) and tigacycline (8.0% resistance rate). A total of 105 (80.2%) CRKP strains carried the Klebsiella pneumoniae carbapenemase (KPC) resistance gene, 15 (11.4%) strains carried the New Delhi Metallo-β-lactamase (NDM) gene, and 4 (3.1%) isolates carried both KPC and NDM genes. Sequence typing (ST) 11 (74.0%) was the dominant sequence type. High detection rates for mrkD (96.2%), fimH (98.5%), entB (100%), and other virulence genes were reported. One hypervirulent CRKP strain was detected. The seven strains of CRKP that did not produce carbapenemase were shown to carry ESBL or AmpC genes and had anomalies in membrane porins OMPK35 and OMPK36, according to whole genome sequencing.Conclusion:In a large-scale tertiary-care general hospital, CRKP mainly carries the KPC gene, has a high drug resistance rate to a variety of antibiotics, and possesses multiple virulence genes. Attention should be paid to CRKP strains with high virulence.

Nocardiosis, despite its rarity and underreporting, is significant due to its severe impact, characterized by high morbidity and mortality rates. The development of a precise, reliable, rapid, and straightforward technique for identifying the pathogenic agent in clinical specimens is crucial to reduce fatality rates and facilitate timely antimicrobial treatment. In this study, we aimed to identify Nocardia spp. in clinical isolates, using MALDI-TOF MS as the primary method, with molecular methods as the gold standard. Clinical Nocardia isolates were identified using 16S rRNA/hsp65/gyrB/secA1/rpoB gene sequencing. Identification performance of the Bruker MALDI Biotyper 3.1 (V09.0.0.0_8468) and MBT Compass 4.1 (V11.0.0.0_10833) for Nocardia identification was evaluated. Seventy-six Nocardia isolates were classified into 12 species through gene sequencing. The MALDI Biotyper 3.1 (V09.0.0.0_8468) achieved 100

OBJECTIVES:To investigate the relationship between the virulence and the carbapenem resistance phenotype of Klebsiella pneumoniae from blood infection, and to identify carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HVKP)strains. METHODS:A total of 192 Klebsiella pneumoniae strains were isolated from blood culture of patients with bloodstream infections from 2016 to 2019, of which 96 isolates were carbapenem-resistant Klebsiella pneumoniae (CRKP) and 96 were carbapenem-sensitive Klebsiella pneumoniae (CSKP). The drug susceptibility was detected by VITEK-2 automatic microbial analyzer; carbapenemase genes, virulence genes and capsule typing were detected by polymerase chain reaction; the high viscosity phenotype of strains was detected by string test, and the genome characteristics of CR-HVKP were detected by whole genome sequencing. Serum killing and biofilm formation test were used to further verify the virulence of CR-HVKP. RESULTS:There were significant differences in drug resistance to common antibiotics, except for minocycline between CSKP and CRKP isolates (all P<0.05). 92 out of 96 CRKP isolates carried carbapenemase genes, mainly blaKPC-2. The string tests were positive in 4 isolates of CRKP and 36 isolates of CSKP (P<0.05). The detection rates of virulence genes Kfu, aerobictin, iutA, ybtS, rmpA, magA, allS, and capsule antigen K1 and K2 in CSKP group were significantly higher than those in CRKP group (all P<0.05). One HVKP strain was detected in the CRKP group (CR-HVKP) and 36 HVKP was detected in the CSKP group (P<0.05). The CR-HVKP strain belonged to the MLST412, serotype K57, expressed iutA, entB, mrkD, fimH, and rmpA virulence genes, and showed strong biofilm formation and significantly increased serum resistance. Whole genome sequencing results showed that this CR-HVKP isolate carried blaSHV-145, blaTEM-1, blaCTX-M-3, fosA6, oqxA5, oqxB26, and aac(3)-IId resistance genes, accompanied by abnormalities in outer membrane protein K (OmpK) 35 and OmpK36. CONCLUSIONS:The drug resistance of CRKP is significantly higher than that of CSKP, while CRKP carrying fewer virulence genes in both number and types compared to CSKP. A new MLST type of carbapenem-resistant and hypervirulent Klebsiella pneumoniae strain has been detected, which requires clinical awareness and epidemiological monitoring.

We aim to validate and evaluate a new rapid and simplified method, called Blood-rsCDM, for the detection and characterization of carbapenemase using 3-aminophenylboronic acid (APBA) and ethylenediaminetetraacetic acid (EDTA) β-lactamase inhibitors from positive blood cultures. We utilized a panel of 172 Enterobacterales strains, including blaKPC (77), blaNDM (48), blaIMP (9), blaVIM (2), blaOXA-181 (2), blaKPC and blaNDM (6), as well as 28 carbapenem-susceptible Enterobacterales isolates, to assess the performance of Blood-rsCDM and the EDTA-carbapenem inactivation method (eCIM). Carbapenemase class was determined using specific inhibitors at 4 h and 6 h by Blood-rsCDM. Blood-rsCDM exhibited a sensitivity of 97.9

Abstract Background Elizabethkingia is emerging as an opportunistic pathogen in humans. The aim of this study was to investigate the clinical epidemiology, antimicrobial susceptibility, virulence factors, and genome features of Elizabethkingia spp. Methods Clinical data from 71 patients who were diagnosed with Elizabethkingia-induced pneumonia and bacteremia between August 2019 and September 2021 were analyzed. Whole-genome sequencing was performed on seven isolates, and the results were compared with a dataset of 83 available Elizabethkingia genomes. Genomic features, Kyoto Encyclopedia of Genes and Genomes (KEGG) results and clusters of orthologous groups (COGs) were analyzed. Results The mean age of the patients was 56.9 ± 20.7 years, and the in-hospital mortality rate was 29.6% (21/71). Elizabethkingia strains were obtained mainly from intensive care units (36.6%, 26/71) and emergency departments (32.4%, 23/71). The majority of the strains were isolated from respiratory tract specimens (85.9%, 61/71). All patients had a history of broad-spectrum antimicrobial exposure. Hospitalization for invasive mechanical ventilation or catheter insertion was found to be a risk factor for infection. The isolates displayed a high rate of resistance to cephalosporins and carbapenems, but all were susceptible to minocycline and colistin. Genomic analysis identified five β-lactamase genes (blaGOB, blaBlaB, blaCME, blaOXA, and blaTEM) responsible for β-lactam resistance and virulence genes involved in stress adaptation (ureB/G, katA/B, and clpP), adherence (groEL, tufA, and htpB) and immune modulation (gmd, tviB, cps4J, wbtIL, cap8E/D/G, and rfbC). Functional analysis of the COGs revealed that “metabolism” constituted the largest category within the core genome, while “information storage and processing” was predominant in both the accessory and unique genomes. The unique genes in our 7 strains were mostly enriched in KEGG pathways related to microRNAs in cancer, drug resistance (β-lactam and vancomycin), ABC transporters, biological metabolism and biosynthesis, and nucleotide excision repair mechanisms. Conclusion The Elizabethkingia genus exhibits multidrug resistance and carries carbapenemase genes. This study presents a comparative genomic analysis of Elizabethkingia, providing knowledge that facilitates a better understanding of this microorganism.

The aim of this study was to comprehensively evaluate metagenomic next-generation sequencing (mNGS), Acid-fast bacillus stain (AFB), MGIT960 culture, polymerase-chain-reaction (PCR), and Xpert MTB/RIF in the diagnosis of tuberculous meningitis (TBM). A cohort of 280 patients who presented with suspected TBM (ie, headache or altered mental status with clinical signs of meningism) were analyzed. The sensitivities of the 5 assays for the diagnosis of TBM ranged from 10.0% to 70.0%. The AFB had the lowest sensitivity of 10.0% (0.545.9), while mNGS and PCR had the highest sensitivity, both at 70.0% (35.4-91.9). mNGS demonstrated a distinct advantage in identifying a wider array of pathogens, including viruses, in CSF samples. PCR was a costeffective option with excellent sensitivity and specificity. However, no single method was statistically significantly better than any other in the diagnosis of TBM. New diagnostic techniques are urgently needed for the independent, rapid and accurate detection of Mycobacterium tuberculosis to guide the diagnosis of TBM. & COPY; 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

BACKGROUND:Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has revolutionized microbial identification. However, there is a lack of data on its performance in identifying filamentous fungi. The objective of our study was to evaluate the accuracy of the Autof ms1000 mass spectrometry for identifying filamentous fungi in the clinical microbiology laboratory.RESULTS:Among 106 samples tested using the Autof ms1000 system, 101 (95.28%) were identified at the genus or species level, and 81 (76.41%) were accurately identified at the species level. Additionally, we developed a new rapid formic acid extraction method with simple pretreatment for filamentous fungi that saved time and provided accurate results.CONCLUSIONS:The Autof ms1000 mass spectrometer proved to be a valuable tool for identifying filamentous fungi. However, upgrading the database is recommended for correctly identifying rare strains.

ObjectiveTo compare the consistency and accuracy of a rapid test method and a traditional test method for pathogen identification, antimicrobial susceptibility and carbapenemase type identification of positive blood culture samples.MethodsA total of 51 positive blood culture samples of bloodstream infection (BSI) were collected between March 2022 and May 2022. All samples were found to be "positive for Gram-negative bacilli" according to the blood smear results. The rapid method was adopted to perform rapid antimicrobial susceptibility test (RAST) and analysis of the positive blood culture samples. According to the RAST result interpretation standards, NG-Test® CARBA 5 was used for rapid carbapenemase detection of the imipenem-resistant strains and the results were confirmed by PCR. In addition, mass spectrometry, VITEK 2 Compact drug sensitivity analysis, and carbapenemase type identification were performed with the colonies cultured with positive samples according to the traditional method.ResultsIn the identification of bacteria, the rapid method and the traditional method had 100% consistency rate in the identification results of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. In the antimicrobial susceptibility test, the consistency rate between the results of the two methods was high and the consistency rate for results for susceptibility to imipenem was 100%. In the identification of carbapenemase type, 18 serinase-producing strains and 3 metal-β-lactamase-producing strains of Enterobacterales were detected by the traditional method. With the rapid method, 18 Klebsiella pneumoniae carbapenemase (KPC)-producing strains, 2 New Delhi metallo-betalactamase (NDM)-producing strains, and 1 imipenem enzyme (IMP)-producing strain were identified in the blood culture samples by using a testing kit. Compared with the PCR results, the sensitivity and specificity of the rapid test for determining carbapenemase types were 100%. In this study, we investigated a rapid method for bacteria and carbapenemase type identification of positive blood culture specimens and found that the turnaround time (TAT) of the rapid method was reduced by 1.94 days on average in comparison with the TAT of the traditional method.ConclusionThe rapid method established in the study can effectively shorten the TAT for pathogenic microorganism identification and antimicrobial susceptibility test of blood culture samples, and the joint report of colloidal gold carbapenemase type identification results can provide a reference for clinicians to use antibiotics appropriately and accurately manage multi-drug resistant bacterial infections.

Background: To investigate the genotype and clinical characteristics of carbapenem-resistant Enterobacteriaceae (CRE) strains in southwest China and provide information on the treatment stopping the spread of the infection.Methods: The clinical information of CRE isolates was collected from 19 hospitals in 12 cities across Sichuan Province, China, between June 2018 and April 2019. The isolates were detected by DNA sequencing of genes encoding carbapenem enzymes and multilocus sequence types (MLSTs).Results: A total of 166 nonrepetitive CRE isolates were isolated during the study period from sputum, blood, urine, and other samples. Klebsiella pneumoniae carbapenemase (KPC) was dominant in Klebsiella pneu-moniae (53.9%), followed by New Delhi metallo-beta-lactamase (NDM) (42.1%). A total of 43 STs were detected. The most common ST of K. pneumoniae was ST11, and that of Escherichia coli was ST410. Pairwise single nucleotide polymorphism (SNP) distances and the likelihood of local transmission by epidemiology were plotted for each species. About 65% of these pairs had <= 20 pairwise SNPs.Conclusion: A large number of CRE strains carried carbapenemase. Although NDM-ST12 K. pneumoniae should not be disregarded, KPC-ST11is the predominant strain. Thus, the possibility of transmission be-tween E. coli and K. pneumoniae could not be ignored.(c) 2022 Published by Elsevier Ltd on behalf of King Saud Bin Abdulaziz University for Health Sciences. CC_BY_4.0

The aim of this study was to investigate the current status of candidemia and evaluate the clinical characteristics, risk factors and outcomes among different species. We conducted a retrospective study by univariate and multivariate analysis between Candida albicans and non-albicans Candida (NAC) species in a Chinese national medical center from 2016 to 2020. Among the 259 episodes, C. albicans (38.6%) was the leading species, followed by C. tropicalis (24.3%), C. parapsilosis (20.5%), and C. glabrata (12.4%). Most C. albicans and C. parapsilosis were susceptible to nine tested antifungal agents, whereas C. tropicalis showed 30.2~65.9% resistance/non-wild-type to four azoles with great cross-resistance, indicating that fluconazole should not be used for empirical antifungal treatment. In multivariable models, the factor related to an increased risk of NAC was glucocorticoid exposure, whereas gastrointestinal hemorrhage and thoracoabdominal drainage catheters were associated with an increased risk in C. albicans. Subgroup analysis revealed leukemia and lymphoma, as well as glucocorticoid exposure, to be factors independently associated with C. tropicalis in comparison with C. albicans candidemia. No significant differences in 7-day mortality or 30-day mortality were observed between C. albicans and NAC. This study may provide useful information with respect to choosing empirical antifungal agents and exploring differences in molecular mechanisms.