Multicentre evaluation of in vitro activity of contezolid against drug-resistant Staphylococcus and Enterococcus.

BACKGROUND: To investigate susceptibility to contezolid, a novel oxazolidinone, multicentre surveillance was conducted involving 2449 strains of Staphylococcus and Enterococcus collected from 65 hospitals across China. METHODS: The MICs of contezolid, linezolid and other clinically significant antibiotics were determined by the broth microdilution method. Consistency with the broth microdilution method for contezolid was assessed using agar dilution method, as well as disc diffusion and ETEST for linezolid, respectively. WGS was conducted on all 20 linezolid-resistant and 30 randomly non-resistant strains to analyse linezolid resistance genes (optrA, poxtA, cfr) and 23S rRNA mutation sites. RESULTS: All strains exhibited WT susceptibility to contezolid, while resistance proportions to daptomycin, vancomycin, teicoplanin, tigecycline and eravacycline ranged from 0% to 5.2% in Staphylococcus, and from 0% to 7.8% in Enterococcus. Linezolid resistance was higher in Enterococcus faecalis (4.4%) compared with Enterococcus faecium (0.2%). Contezolid showed a lower MIC50 (0.5 mg/L) than linezolid (2 mg/L) for methicillin-resistant Staphylococcus. Against Enterococcus, contezolid demonstrated a cumulative MIC percentage of 70% for VRE and 39.1% for E. faecalis (at MIC = 1 mg/L), whereas linezolid showed 0% and 1.1%, respectively. Among the 20 linezolid-resistant Enterococcus strains, all carried the optrA gene without 23S rRNA mutations. For contezolid, MICs were 4 mg/L for 19 strains and 2 mg/L for 1 strain. The ETEST, agar dilution and disc diffusion methods showed essential and categorical agreements of >90% for linezolid, with no major errors or very major errors. CONCLUSIONS: Contezolid demonstrated significant in vitro antibacterial activity against methicillin-resistant Staphylococcus, VRE and linezolid-resistant E. faecalis.

Establishment of epidemiological cut-off values for eravacycline, against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii and Staphylococcus aureus.

OBJECTIVES: To establish the epidemiology cut-off (ECOFF) values of eravacycline against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Acinetobacter baumannii and Staphylococcus aureus, from a multi-centre study in China. METHODS: We collected 2500 clinical isolates from five hospitals in China from 2017 to 2020. The MICs of eravacycline were determined using broth microdilution. The ECOFF values of eravacycline against the five species commonly causing cIAIs were calculated using visual estimation and ECOFFinder following the EUCAST guideline. RESULTS: The MICs of eravacycline against all the strains were in the range of 0.004-16 mg/L. The ECOFF values of eravacycline were 0.5 mg/L for E. coli, 2 mg/L for K. pneumonia and E. cloacae, and 0.25 mg/L for A. baumannii and S. aureus, consistent with the newest EUCAST publication of eravacycline ECOFF values for the populations. No discrepancy was found between the visually estimated and 99.00% ECOFF values calculated using ECOFFinder. CONCLUSIONS: The determined ECOFF values of eravacycline against the five species can assist in distinguishing wild-type from non-wild-type strains. Given its promising activity, eravacycline may represent a member of the tetracycline class in treating cIAIs caused by commonly encountered Gram-negative and Gram-positive pathogens.

Genomic epidemiology reveals multiple mechanisms of linezolid resistance in clinical enterococci in China.

BACKGROUND: Infections caused by linezolid-resistant enterococci (LRE) are clinically difficult to treat and threaten patient health. However, there is a lack of studies on long time-span LRE strains in China. For this reason, our study comprehensively revealed the resistance mechanisms of LRE strains collected in a Chinese tertiary care hospital from 2011 to 2022. METHODS: Enterococcal strains were screened and verified after retrospective analysis of microbial data. Subsequently, 65 LRE strains (61 Enterococcus faecalis and 4 Enterococcus faecium, MIC ≥ 8 µg/ml), 1 linezolid-intermediate Enterococcus faecium (MIC = 4 µg/ml) and 1 linezolid-susceptible Enterococcus faecium (MIC = 1.5 µg/ml) were submitted for whole-genome sequencing (WGS) analysis and bioinformatics analysis. RESULTS: The optrA gene was found to be the most common linezolid resistance mechanism in our study. We identified the wild-type OptrA and various OptrA variants in 98.5% of LRE strains (61 Enterococcus faecalis and 3 Enterococcus faecium). We also found one linezolid-resistant Enterococcus faecium strain carried both optrA and cfr(D) gene, while one linezolid-resistant Enterococcus faecium only harbored the poxtA gene. Most optrA genes (55/64) were located on plasmids, with impB-fexA-optrA, impB-fexA-optrA-erm(A), fexA-optrA-erm(A), and fexA-optrA segments. A minority of optrA genes (9/64) were found on chromosomes with the Tn6674-like platform. Besides, other possible linezolid resistance-associated mechanisms (mutations in the rplC and rplD genes) were also found in 26 enterococcal strains. CONCLUSIONS: Our study suggested that multiple mechanisms of linezolid resistance exist among clinical LRE strains in China.

Clonal Dissemination of Antifungal-Resistant Candida haemulonii, China.

Candida haemulonii, a relative of C. auris, frequently shows antifungal resistance and is transmissible. However, molecular tools for genotyping and investigating outbreaks are not yet established. We performed genome-based population analysis on 94 C. haemulonii strains, including 58 isolates from China and 36 other published strains. Phylogenetic analysis revealed that C. haemulonii can be divided into 4 clades. Clade 1 comprised strains from China and other global strains; clades 2-4 contained only isolates from China, were more recently evolved, and showed higher antifungal resistance. Four regional epidemic clusters (A, B, C, and D) were identified in China, each comprising ≥5 cases (largest intracluster pairwise single-nucleotide polymorphism differences <50 bp). Cluster A was identified in 2 hospitals located in the same city, suggesting potential intracity transmissions. Cluster D was resistant to 3 classes of antifungals. The emergence of more resistant phylogenetic clades and regional dissemination of antifungal-resistant C. haemulonii warrants further monitoring.

Enhanced Antibacterial Ability of Electrospun PCL Scaffolds Incorporating ZnO Nanowires.

The infection of implanted biomaterial scaffolds presents a major challenge. Existing therapeutic solutions, such as antibiotic treatment and silver nanoparticle-containing scaffolds are becoming increasingly impractical because of the growth of antibiotic resistance and the toxicity of silver nanoparticles. We present here a novel concept to overcome these limitations, an electrospun polycaprolactone (PCL) scaffold functionalised with zinc oxide nanowires (ZnO NWs). This study assessed the antibacterial capabilities and biocompatibility of PCL/ZnO scaffolds. The fabricated scaffolds were characterised by SEM and EDX, which showed that the ZnO NWs were successfully incorporated and distributed in the electrospun PCL scaffolds. The antibacterial properties were investigated by co-culturing PCL/ZnO scaffolds with Staphylococcus aureus. Bacterial colonisation was reduced to 51.3% compared to a PCL-only scaffold. The biocompatibility of the PCL/ZnO scaffolds was assessed by culturing them with HaCaT cells. The PCL scaffolds exhibited no changes in cell metabolic activity with the addition of the ZnO nanowires. The antibacterial and biocompatibility properties make PCL/ZnO a good choice for implanted scaffolds, and this work lays a foundation for ZnO NWs-infused PCL scaffolds in the potential clinical application of tissue engineering.

Optimized Sequencing Adaptors Enable Rapid and Real-Time Metagenomic Identification of Pathogens during Runtime of Sequencing.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) offers the promise of unbiased detection of emerging pathogens. However, in indexed sequencing, the sequential paradigm of data acquisition, demultiplexing, and analysis restrain read assignment in advance and real-time analysis, resulting in lengthy turnaround time for clinical metagenomic detection. METHODS: We described the utility of internal-index adaptors with different lengths of barcode in multiplex sequencing. The base composition for each position within these adaptors was well-balanced to ensure nucleotide diversity and optimal sequencing performance and to achieve the early assignment of reads by first sequencing the barcodes. Combined with an automated library preparation device, we delivered a rapid and real-time bioinformatics pathogen identification solution for the Illumina NextSeq platform. The diagnostic performance was evaluated by testing 153 lower respiratory tract specimens using mNGS in comparison to culture, 16S/internal transcribed spacer amplicon sequencing, and additional PCR-based tests. RESULTS: By calculating the average F1 scores of all read lengths under different threshold values, we established the optimal threshold for pathogens identification, and found that 36 bp was the optimal shortest read length for rapid mNGS analysis. Rapid detection had a negative percentage agreement and positive percentage agreement of 100% and 85.1% for bacteria and 97.4% and 80.3% for fungi, when compared to a composite standard. The rapid mNGS solution enabled accurate pathogen identification in about 9.1 to 10.1 h sample-to-answer turnaround time. CONCLUSIONS: Optimized internal index adaptors combined with a real-time analysis pipeline provide a potential tool for a first-line test in critically ill patients.

Diagnosis of Coxiella burnetii infection via metagenomic next-generation sequencing: a case report.

BACKGROUND: Coxiella burnetii, the etiologic agent of Q fever, is mainly responsible for endocardite. But there are only a few cases of Coxiella burnetii-caused wound infection have been published, because the pathogen is very difficult to isolate using conventional culture methods. CASE PRESENTATIONS: A 76-year-old man, underwent endovascular repair of ruptured left iliac aneurysm plus abdominal aortic aneurysm under general anesthesia in 2018. Left iliac fossa mass resection was performed in 2020. After operation, the wound in the left iliac fossa was repeatedly ruptured and not healing. We used the wound tissue to perform the Metagenomics next-generation sequencing (mNGS), Coxiella burnetii was detected. Sanger sequencing and serologic verification of Coxiella burnetii all showed positive results. CONCLUSIONS: This study proved that mNGS was an effective method to detect clinically unexplained infections, and showed the ability of pathogen identification with high sensitivity and accuracy.

The seroprevalence and kinetics of IgM and IgG in the progression of COVID-19.

BACKGROUND: SARS-CoV-2 is a novel coronavirus first recognized in late December 2019 that causes coronavirus disease 19 (COVID-19). Due to the highly contagious nature of SARS-CoV-2, it has developed into a global pandemic in just a few months. Antibody testing is an effective method to supplement the diagnosis of COVID-19. However, multicentre studies are lacking to support the understanding of the seroprevalence and kinetics of SARS-CoV-2 antibodies in COVID-19 epidemic regions. METHOD: A multicentre cross-sectional study of suspected and confirmed patients from 4 epidemic cities in China and a cohort study of consecutive follow-up patients were conducted from 29/01/2020 to 12/03/2020. IgM and IgG antibodies elicited by SARS-CoV-2 were tested by a chemiluminescence assay. Clinical information, including basic demographic data, clinical classification, and time interval from onset to sampling, was collected from each centre. RESULTS: A total of 571 patients were enrolled in the cross-sectional study, including 235 COVID-19 patients and 336 suspected patients, each with 91.9%:2.1% seroprevalence of SARS-CoV-2 IgG and 92.3%:5.4% seroprevalence of SARS-CoV-2 IgM. The seroprevalence of SARS-CoV-2 IgM and IgG in COVID-19 patients was over 70% less than 7 days after symptom onset. Thirty COVID-19 patients were enrolled in the cohort study and followed up for 20 days. The peak concentrations of IgM and IgG were reached on the 10th and 20th days, respectively, after symptom onset. The seroprevalence of COVID-19 IgG and IgM increased along with the clinical classification and treatment time delay. CONCLUSION: We demonstrated the kinetics of IgM and IgG SARS-CoV-2 antibodies in COVID-19 patients and the association between clinical classification and antibodies, which will contribute to the interpretation of IgM and IgG SARS-CoV-2 antibody tests and in predicting the outcomes of patients with COVID-19.

Establishment of epidemiological cut-off values for cefoselis, a new fourth-generation cephalosporin, against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Proteus mirabilis and Pseudomonas aeruginosa.

OBJECTIVES: To establish the epidemiological cut-off values (ECOFFs) for cefoselis against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Proteus mirabilis and Pseudomonas aeruginosa. METHODS: We collected 2288 non-repetitive clinical isolates from five laboratories throughout four cities in China. The cefoselis MICs and inhibition zone diameters for all isolates were established using the broth microdilution method and the disc diffusion method following EUCAST guidelines. MIC ECOFFs were determined by visual estimation and ECOFFinder software. Zone diameter ECOFFs were set if a high correlation of MICs and inhibition zone diameters was found by Pearson correlation. Zone diameter ECOFFs were finally determined by the visual estimate method. RESULTS: MICs of cefoselis were distributed from 0.008 to >256 mg/L for the four Enterobacterales species and from 0.25 to >256 mg/L for P. aeruginosa. MIC ECOFFs were 0.125 mg/L for E. coli, K. pneumoniae and P. mirabilis, 0.25 mg/L for E. cloacae and 32 mg/L for P. aeruginosa. A high correlation of MICs and zone diameters was observed for all Enterobacterales (|r| > 0.8, P < 0.001) and a relatively high correlation was found for P. aeruginosa (|r| = 0.71, P < 0.001). The zone diameter ECOFF was 24 mm for E. cloacae, E. coli and K. pneumoniae, 26 mm for P. mirabilis and 21 mm for P. aeruginosa. CONCLUSIONS: We determined MIC and zone diameter ECOFFs for cefoselis against four Enterobacterales species and P. aeruginosa. The establishment of ECOFFs for cefoselis provides clinicians with helpful guidance to differentiate WT and non-WT pathogens.

Determination of norvancomycin epidemiological cut-off values (ECOFFs) for Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus hominis.

OBJECTIVES: To determine the epidemiological cut-off values (ECOFFs) of norvancomycin for Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus hominis. METHODS: We collected 1199 clinical isolates of Staphylococcus species from five laboratories located in four cities in China. MICs and inhibitory zone diameters of norvancomycin were determined by broth microdilution and the disc diffusion method, separately. ECOFFs of norvancomycin for four species were calculated by ECOFFinder software following EUCAST principles. Methicillin and vancomycin resistance genes (mecA/mecC and vanA/vanB/vanC/vanD/vanE) were screened for by PCR in all isolates. Pearson correlation and χ2 test were used to calculate the correlation of MICs and inhibition zone diameters, and MICs and resistance genes, respectively. RESULTS: MICs of norvancomycin for all strains from five laboratories fell in the range of 0.12-2 mg/L. ECOFFs of norvancomycin were determined to be 2 mg/L for S. epidermidis and S. haemolyticus and 1 mg/L for S. aureus and S. hominis. A weak correlation was observed between MIC values and zone diameters for S. haemolyticus (r = -0.36) and S. hominis (r = -0.26), while no correlation was found for S. epidermidis and S. aureus. The mecA gene was detected in 63.1% of Staphylococcus, whereas no isolate carried mecC, vanA, vanB, vanC, vanD or vanE. ECOFFs of norvancomycin were not correlated with mecA gene carriage in Staphylococcus species. CONCLUSIONS: ECOFFs of norvancomycin for four Staphylococcus species were determined, which will be helpful to differentiate WT strains. The correlation of MICs and zone diameters of norvancomycin was weak in Staphylococcus species.

Distribution and Antifungal Susceptibility of Candida Species Causing Candidemia in China: An Update From the CHIF-NET Study.

BACKGROUND: Candidemia is the most common, serious fungal infection and Candida antifungal resistance is a challenge. We report recent surveillance of candidemia in China. METHODS: The study encompassed 77 Chinese hospitals over 3 years. Identification of Candida species was by mass spectrometry and DNA sequencing. Antifungal susceptibility was determined using the Clinical and Laboratory Standards Institute broth microdilution method. RESULTS: In total, 4010 isolates were collected from candidemia patients. Although C. albicans was the most common species, non-albicans Candida species accounted for over two-thirds of isolates, predominated C. parapsilosis complex (27.1%), C. tropicalis (18.7%), and C. glabrata complex (12.0%). Most C. albicans and C. parapsilosis complex isolates were susceptible to all antifungal agents (resistance rate <5%). However, there was a decrease in voriconazole susceptibility to C. glabrata sensu stricto over the 3 years and fluconazole resistance rate in C. tropicalis tripled. Amongst less common Candida species, over one-third of C. pelliculosa isolates were coresistant to fluconazole and 5-flucytocine, and >56% of C. haemulonii isolates were multidrug resistance. CONCLUSIONS: Non-albicans Candida species are the predominant cause of candidemia in China. Azole resistance is notable amongst C. tropicalis and C. glabrata. Coresistance and multidrug resistance has emerged in less common Candida species.

In vitro activity of sulbactam/durlobactam against clinical isolates of Acinetobacter baumannii collected in China.

BACKGROUND: Durlobactam is a broad-spectrum inhibitor of class A, C and D ß-lactamases. Sulbactam is a generic ß-lactam most commonly used as a ß-lactamase inhibitor in combination with ampicillin; however, it has a unique property in that it has selective intrinsic activity against Acinetobacter baumannii. Currently, there is widespread resistance caused by multiple ß-lactamases including class A carbapenemases and class C and class D enzymes. The addition of durlobactam to sulbactam restores in vitro activity against MDR A. baumannii that possess multiple ß-lactamases. OBJECTIVES: Previously, susceptibility data for sulbactam/durlobactam were limited to isolates from patients in Western countries. This study was undertaken to determine the activity of sulbactam/durlobactam against A. baumannii isolated from patients in mainland China. METHODS: Nine hundred and eighty-two recent A. baumannii clinical isolates were collected from 22 sites across mainland China during 2016-18. The isolates were collected from lower respiratory tract, intra-abdominal, urinary tract and skin and skin structure infections. The in vitro activities of sulbactam/durlobactam and comparators were determined by broth microdilution. RESULTS: The addition of durlobactam restored the activity of sulbactam against the majority of the strains tested. The MIC90 of sulbactam/durlobactam was 2 mg/L for all A. baumannii, compared with 64 mg/L for sulbactam alone. The MIC90 of sulbactam/durlobactam of 2 mg/L remained unchanged for 831 carbapenem-resistant isolates. Colistin was the only comparator with comparable activity (MIC90 = 1 mg/L). CONCLUSIONS: This study demonstrated the potential utility of sulbactam/durlobactam for the treatment of infections caused by A. baumannii in China.

Synthesis of urchin-like NiCo2S4 electrode materials based on a two-step hydrothermal method for high-capacitance supercapacitors.

Transition metal sulfides have been considered as promising electrode materials for future super-capacitors due to their spinel structures and environmentally friendly properties. Among these materials, NiCo2S4 compounds exhibit high theoretical specific capacity but poor cycling performance. To address this issue, we synthesize several NiCo2S4 urchin balls. The NCS-1.5 nanospheres demonstrate a specific capacitance of 1352.2 F g-1 at a current density of 1 A g-1, and maintain high specific capacity after 10 000 charge-discharge cycles. An asymmetric capacitor assembled with the NCS-1.5 sample as the cathode and activated carbon as the anode achieve an energy density of 45.5 W h kg-1 at 2025 W kg-1. The urchin-like nanospheres also facilitate the combination with other materials, providing potential insights for the synthesis of supercapacitor electrode materials.

Effect of ceftazidime-avibactam combined with different antimicrobials against carbapenem-resistant Klebsiella pneumoniae.

This study aimed to assess the in vitro efficacy of ceftazidime-avibactam (CZA) in combination with various antimicrobial agents against carbapenem-resistant Klebsiella pneumoniae (CRKP). We selected 59 clinical CRKP isolates containing distinct drug resistance mechanisms. The minimum inhibitory concentrations (MICs) of meropenem (MEM), colistin (COL), eravacycline (ERA), amikacin (AK), fosfomycin (FOS), and aztreonam (ATM), both individually and in combination with CZA, were tested using the checkerboard method. The interactions of antimicrobial agent combinations were assessed by fractional inhibitory concentration index (FICI) and susceptible breakpoint index (SBPI). The time-kill curve assay was employed to dynamically evaluate the effects of these drugs alone and in combination format. In the checkerboard assay, the combination of CZA+MEM showed the highest level of synergistic effect against both KPC-producing and carbapenemase-non-producing isolates, with synergy rates of 91.3% and 100%, respectively. Following closely was the combination of FOS+CZA . For metallo-beta-lactamases (MBLs) producing strains, ATM+CZA displayed complete synergy, while the combination of MEM+CZA showed a synergy rate of only 57.14% for NDM-producing strains and 91.67% for IMP-producing strains. In the time-kill assay, MEM+CZA also demonstrated significant synergistic effects against the two KPC-2-producing isolates (Y070 and L70), the two carbapenemase-non-producing isolates (Y083 and L093), and the NDM-1-producing strain L13, with reductions in log10 CFU/mL exceeding 10 compared to the control. Against the IMP-producing strain Y047, ATM+CZA exhibited the highest synergistic effect, resulting in a log10 CFU/mL reduction of 10.43 compared to the control. The combination of CZA and MEM exhibited good synergistic effects against KPC-producing and non-enzyme-producing strains, followed by the FOS+CZA combination. Among MBL-producing strains, ATM+CZA demonstrated the most pronounced synergistic effect. However, the combinations of CZA with ERA, AK, and COL show irrelevant effects against the tested clinical isolates. IMPORTANCE: Our study confirmed the efficacy of the combination CZA+MEM against KPC-producing and non-carbapenemase-producing strains. For metalloenzyme-producing strains, CZA+ATM demonstrated the most significant synergy. Additionally, CZA exhibited a notable synergy effect when combined with FOS. These combination therapies present promising new options for the treatment of CRKP infection.

In-vitro activities of essential antimicrobial agents including aztreonam/avibactam, eravacycline, colistin and other comparators against carbapenem-resistant bacteria with different carbapenemase genes: A multi-centre study in China, 2021.

OBJECTIVE: Carbapenem-resistant bacteria (CRB), including carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA) and carbapenem-resistant Enterobacterales (CRE), pose a considerable threat to public health in China. Eravacycline, aztreonam/avibactam and colistin are important antimicrobial agents for the treatment of serious infections caused by CRB. This study aimed to evaluate the prevalence of CRB strains, and the susceptibility of commonly used clinical antimicrobial agents against strains with different carbapenemase genes. METHODS: In total, 7194 gram-negative bacteria strains were collected from different regions of China, and 924 carbapenem-resistant strains were identified. All strains were from confirmed infections. Antimicrobial susceptibility testing, covering 21 antimicrobial agents including aztreonam/avibactam, eravacycline, colistin and other comparators, was performed using the broth microdilution method. Carbapenemase genes (blaKPC, blaNDM, blaOXA, blaIMP and blaVIM) were screened using polymerase chain reaction amplification and sequence analysis. All statistical analyses were performed using Statistical Package for the Social Sciences Version 23.0. RESULTS: The isolation rates of CRE, CRAB and CRPA were 6.31% (332/5265), 62.95% (440/699) and 15.20% (152/1000), respectively. The predominant carbapenemase in carbapenem-resistant Escherichia coli (CRECO) was NDM, while in carbapenem-resistant Klebsiella pneumoniae (CRKP), it was KPC. All CRAB produced OXA-23, and 85.52% of CRPA did not produce any of the following carbapenemases: NDM, KPC, VIM, IMP and OXA. Aztreonam/avibactam, colistin and eravacycline exhibited high antimicrobial activity against different species producing various carbapenemases. Compared with ceftazidime/avibactam, aztreonam/avibactam demonstrated superior antimicrobial activity, particularly pronounced in CRECO and strains producing metallo-beta-lactamases. In comparisons between tigecycline and eravacycline, the latter maintained higher antimicrobial activity across different species. Antimicrobial agents exhibited varying levels of activity against strains with different resistance mechanisms. CONCLUSIONS: Using aztreonam/avibactam, eravacycline and colistin to treat infections caused by CRB offers significant advantages. These findings will guide clinical practice and optimize antimicrobial administration.

Emergence of ST463 exoU-Positive, Imipenem-Nonsusceptible Pseudomonas aeruginosa Isolates in China.

This study investigated the resistance mechanisms and the distribution and proportions of virulence genes, including exoU, in 182 imipenem-nonsusceptible Pseudomonas aeruginosa (INS-PA) strains collected from China in 2019. There was no obvious prevalent sequence type or concentrated evolutionary multilocus sequence typing (MLST) type on the INS-PA phylogenetic tree in China. All of the INS-PA isolates harbored ß-lactamases with/without other antimicrobial mechanisms, such as gross disruption of oprD and overexpression of efflux genes. Compared with exoU-negative isolates, exoU-positive isolates (25.3%, 46/182) presented higher virulence in A549 cell cytotoxicity assays. The southeast region of China had the highest proportion (52.2%, 24/46) of exoU-positive strains. The most frequent exoU-positive strains belonged to sequence type 463 (ST463) (23.9%, 11/46) and presented multiple resistance mechanisms and higher virulence in the Galleria mellonella infection model. The complex resistance mechanisms in INS-PA and the emergence of ST463 exoU-positive, multidrug-resistant P. aeruginosa strains in southeast China indicated a challenge that might lead to clinical treatment failure and higher mortality. IMPORTANCE This study investigates the resistance mechanisms and distribution and proportions of virulence genes of imipenem-nonsusceptible Pseudomonas aeruginosa (INS-PA) isolates in China in 2019. Harboring PDC and OXA-50-like genes is discovered as the most prevalent resistance mechanism in INS-PA, and the virulence of exoU-positive INS-PA isolates was significantly higher than that of exoU-negative INS-PA isolates. There was an emergence of ST463 exoU-positive INS-PA isolates in Zhejiang, China, most of which presented multidrug resistance and hypervirulence.

Rapid automated antifungal susceptibility testing system for yeasts based on growth characteristics.

Fungal pathogens are a major threat to public health, as they are becoming increasingly common and resistant to treatment, with only four classes of antifungal medicines currently available and few candidates in the clinical development pipeline. Most fungal pathogens lack rapid and sensitive diagnostic techniques, and those that exist are not widely available or affordable. In this study, we introduce a novel automated antifungal susceptibility testing system, Droplet 48, which detects the fluorescence of microdilution wells in real time and fits growth characteristics using fluorescence intensity over time. We concluded that all reportable ranges of Droplet 48 were appropriate for clinical fungal isolates in China. Reproducibility within ±2 two-fold dilutions was 100%. Considering the Sensititre YeastOne Colorimetric Broth method as a comparator method, eight antifungal agents (fluconazole, itraconazole, voriconazole, caspofungin, micafungin, anidulafungin, amphotericin B, and 5-flucytosine) showed an essential agreement of >90%, except for posaconazole (86.62%). Category agreement of four antifungal agents (fluconazole, caspofungin, micafungin, and anidulafungin) was >90%, except for voriconazole (87.93% agreement). Two Candida albicans isolates and anidulafungin showed a major discrepancy (MD) (2.60%), and no other MD or very MD agents were found. Therefore, Droplet 48 can be considered as an optional method that is more automated and can obtain results and interpretations faster than previous methods. However, the optimization of the detection performance of posaconazole and voriconazole and promotion of Droplet 48 in clinical microbiology laboratories still require further research involving more clinical isolates in the future.

Background Filtering of Clinical Metagenomic Sequencing with a Library Concentration-Normalized Model.

Metagenomic next-generation sequencing (mNGS) can accurately detect pathogens in clinical samples. However, wet-lab contamination constrains mNGS analysis and may result in erroneous interpretation of results. Many existing methods rely on large-scale observational microbiome studies and may not be applicable to clinical mNGS tests. By generation of a pretrained profile of common laboratory contaminants, we developed an mNGS noise-filtering model based on the inverse linear relationship between microbial sequencing reads and sample library concentration, named the background elimination and correction by library concentration-normalized (BECLEAN) model. Its efficacy was evaluated with bacteria- and yeast-spiked samples and 28 cerebrospinal fluid (CSF) specimens. The diagnostic accuracy, precision, sensitivity, and specificity of BECLEAN with reference to conventional methods and diagnosis were 92.9%, 86.7%, 100%, and 86.7%, respectively. BECLEAN led to a dramatic reduction of background noise without affecting the true-positive rate and thus can provide a time-saving and convenient tool in various clinical settings. IMPORTANCE Most of the existing methods to remove wet-lab contamination rely on large-scale observational microbiome studies and may not be applicable to clinical mNGS testing in individual cases. In clinical settings, only a handful of samples might be sequenced in a run. The lab-specific microbiome can complicate existing statistical approaches for removing contamination from small-scale clinical metagenomic sequencing data sets; thus, use of a preliminary lab-specific training set is necessary. Our study provides a rapid and accurate background-filtering tool for clinical metagenomic sequencing by generation of a pretrained profile of common laboratory contaminants. Notably, our work demonstrates that the inverse linear relationship between microbial sequencing reads and library concentration can serve to identify true contaminants and evaluate the relative abundance of a taxon in samples by comparing the observed microbial reads to the model-predicted value. Our findings extend the previously published research and demonstrate confirmatory results in clinical settings.

In vitro activity of lactone ketolide nafithromycin (WCK 4873) against Streptococcus pneumoniae isolates enriched with macrolide-resistance phenotype collected from mainland China.

Background: Widespread MDR Streptococcus pneumoniae in China translates clinically into a substantial pneumococcal disease burden and related morbidity and mortality, particularly in the elderly and children. Nafithromycin (WCK 4873), a novel lactone ketolide class of antibiotic designed with a 3 day, once-daily regimen is highly active against resistant pneumococci and other community respiratory pathogens. It is currently in clinical development for the treatment of community-acquired bacterial pneumonia (CABP). Objectives: To determine the in vitro activity of nafithromycin against clinical S. pneumoniae isolates collected during 2015-21 from three hospitals in mainland China. Methods: A total of 920 clinical isolates (one isolate per patient), which predominantly with the macrolide- and clindamycin-resistant phenotype were included in this study. The MICs of nafithromycin and other antibiotics tested were determined using the reference broth microdilution method. Results: Clinical S. pneumoniae isolates used in this study showed high macrolide and clindamycin resistance (>95% against erythromycin and azithromycin and 80% against clindamycin) for which nafithromycin showed potent activity (MIC50/90; 0.03/0.06 mg/L) with 100% susceptibility at a proposed pharmacokinetics/pharmacodynamics (PK/PD) breakpoint of 0.25 mg/L. Among other classes of antibiotics tested, moxifloxacin also showed good activity while amoxicillin/clavulanate and ceftriaxone showed lower susceptibility. Conclusions: Nafithromycin exhibited therapeutically relevant in vitro antibacterial activity against contemporary highly resistant pneumococci collected from mainland China. This study supports the clinical development of nafithromycin for the management of CABP caused by pneumococci in China.

Antimicrobial susceptibility to polymyxin B and other comparators against Gram-negative bacteria isolated from bloodstream infections in China: Results from CARVIS-NET program.

Objective: To investigate the bacterial distribution and antimicrobial resistance profile of clinical isolates from Gram-negative bacteria bloodstream infections (GNBSI) in China. Methods: The clinical bacterial strains isolated from blood culture were collected during April 2019 to December 2021 in 21 member hospitals of China Bloodstream Gram-negative Pathogens Antimicrobial Resistance and Virulence Surveillance Network (CARVIS-NET). Antibiotic susceptibility test was conducted by broth microdilution method recommended by Clinical and Laboratory Standards Institute (CLSI, United States). WHONET 2021 and SPSS 22.0 were used to analyze data. Results: During the study period, 1939 Gram-negative bacteria were collected from 21 hospitals, among which 1,724 (88.9%) were Enterobacteriaceae, 207 (10.7%) were non-fermenting Gram-negative bacteria and 8 (0.4%) were others. The top five bacterial species were Escherichia coli (46.2%), Klebsiella pneumoniae (31.6%), Pseudomonas aeruginosa (4.9%), Acinetobacter baumannii (4.2%) and Enterobacter cloacae (3.0%). For K. pneumoniae, antibiotic resistance was mainly prevalent in hospital-associated bloodstream infections, while for A. baumannii, antibiotic resistance was mainly prevalent in community-associated bloodstream infections. It is worth mentioning that 94.1% of the 1939 Gram-negative isolates were susceptible to polymyxin B. The sensitivity of the strains involved in our investigation to polymyxin B is highly correlated with their sensitivity to colistin. Conclusion: The surveillance results in CARVIS-NET-2021 showed that the main pathogens of GNBSI in China were Enterobacteriaceae, while E. coli was the most common pathogen. The resistance rates of K. pneumonia, P. aeruginosa, A. baumannii, and E. cloacae to multiple antibiotics kept on a high level. In many cases, polymyxin B and colistin has become the last-resort agents to combat bloodstream infections caused by multidrug-resistant (MDR) Gram-negative bacteria.