Xiaoli Zhang

The integration of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with machine learning offers clinical microbiology laboratories a way to rapidly obtain information on antimicrobial resistance, typing, and molecular markers beyond routine species identification. However, translation from research to clinical practice remains constrained by three major bottlenecks: substantial heterogeneity in spectral acquisition and preprocessing, the decisive influence of validation design on the strength of evidence, and a systematic lack of workflow evaluation data in implementation studies. Available evidence indicates that culture duration, measurement mode, and cross-center or temporal changes can substantially affect model generalizability. Yet most studies still focus on internal discrimination, with insufficient evaluation of calibration reliability, automated output coverage, confirmatory testing burden, and time gained in reporting; few studies directly link model outputs to antimicrobial adjustment or infection-control actions. This review synthesizes advances across five dimensions: spectrum formation, clinical tasks, validation design, laboratory workflow integration, and postdeployment monitoring. Based on the hierarchy of validation evidence, we propose that the field is moving from feature-association discovery toward validation of clinical transferability. Future priorities should therefore shift from increasingly complex algorithms to standardized multicenter spectrum-sharing and validation frameworks and prospective workflow studies that determine whether additional spectral information can yield measurable time or resource savings.

The Enterobacter cloacae complex (ECC) comprises major nosocomial pathogens and is increasingly resistant to last-line agents such as colistin. Here, we developed a rapid workflow that integrates negative-ion mode MALDI-TOF mass spectrometry with machine learning to identify colistin-resistant ECC (COL-R-ECC). We analyzed 267 clinical ECC isolates; species identification was performed by MALDI-TOF MS and verified by whole-genome sequencing. A one-dimensional convolutional neural network (1D-CNN) with a squeeze-and-excitation (SE) module was trained on spectra from 217 isolates and evaluated on an independent external cohort of 50 isolates. To enhance analytical specificity, we applied a standardized lipid extraction protocol and focused on lipid A–enriched signals (m/z 1500–3000). Spectra were binned at 0.1 Da and smoothed using a Savitzky–Golay filter. The optimized model achieved 95.5% accuracy (AUROC = 0.986; F1 = 0.943) in internal testing and 96.0% accuracy (F1 = 0.960) in external validation, outperforming conventional machine-learning baselines. SHAP analysis highlighted 30 lipid-associated features that contributed most to the predictions, providing interpretable clues to potential resistance mechanisms. Overall, this workflow enables COL-R-ECC identification within ∼1 h from colony processing, substantially faster than standard susceptibility testing, and may support earlier targeted therapy and infection control.

Carbapenem-resistant Enterobacter cloacae complex (CRECC) has become an increasingly important pathogen in healthcare-associated infections, with limited treatment options and a high mortality rate. As reports of antimicrobial resistance continue to rise, tigecycline (TGC) and ceftazidime-avibactam (CZA) have emerged as the last-line therapies for CRECC infections. The aim of this study was to investigate collateral sensitivity to ceftazidime-avibactam following the acquisition of tigecycline resistance in NDM-producing CRECC, and to explore the transcriptional changes associated with this phenomenon and propose potential mechanistic hypotheses. Antimicrobial susceptibility profiles were determined using the broth microdilution method, and changes in colony morphology were analyzed. Transcriptomic sequencing was performed to characterize global gene expression alterations associated with antimicrobial resistance, and an in vivo Galleria mellonella infection model was used to assess the virulence of the mutant strains. Both drug-resistant mutants displayed a stable mucoid phenotype with marked collateral susceptibility to CZA, with minimum inhibitory concentrations decreasing from greater than 128 mg/L to 0.5-1 mg/L. Compared with the parental strains, these mutants showed thickened cell surface structures, impaired growth, reduced serum tolerance, and significantly attenuated virulence in the Galleria mellonella infection model. Transcriptomic analysis revealed marked downregulation of metallo-β-lactamase-related genes at the transcriptional level. In this study of two clinical isolates, collateral sensitivity to CZA was associated with transcriptional downregulation of bla_NDM and altered bacterial metabolism. These findings suggest a potential trade-off between tigecycline resistance and CZA susceptibility that warrants further functional validation.

Utilizing treatment strategies based on collateral sensitivity (CS) represents a promising approach to suppressing antibiotic resistance. Although the mechanism of CS between numerous drugs has been researched, the mechanism of CS between tigecycline and colistin remains unclear. Therefore, the purpose of our research is to investigate the possible mechanism by which tigecycline affects colistin CS in the Enterobacter cloacae complex. Tigecycline induction significantly reduced the minimum inhibitory concentration of carbapenem-resistant Enterobacter cloacae complex (CRECC) to colistin, and sequencing revealed a single-base deletion at the RamR binding site. Complementation experiments demonstrated that deletion of the RamR binding site increased the resistance of CRECC417 to colistin and tigecycline by 2-fold and 4-fold, respectively. Transcriptomic comparison analysis of strains before and after CRECC417 induction revealed a total of 1,977 genes with significant differences in expression. Genes associated with carbohydrate, amino acid, and inorganic ion metabolism were the most highly enriched. Furthermore, the observed increase in colistin susceptibility in CRECC417R can be attributed to the inhibition of quorum sensing and biofilm formation pathways, as well as increased expression levels of genes associated with lipopolysaccharide biosynthesis and modification. IMPORTANCE: Due to the overuse of antibiotics, antimicrobial resistance (AMR) has become a serious threat to global public health. Dosage regimens based on bacterial CS can reduce antibiotic use without reducing efficacy, thereby reducing antibiotic-related toxicity risks, expanding the scope of antibiotic application, and limiting the development of antibiotic resistance. In this study, we analyzed the drug resistance mutations and global transcriptional changes in CRECC after tigecycline induction through genomics and transcriptomics. Our study showed that tigecycline exposure significantly inhibited quorum sensing pathways and biofilm formation. There were significant changes in the transcriptional levels of genes related to cell membrane lipopolysaccharide synthesis and modification, but no mutations were found in genes related to colistin resistance. These findings provide valuable insights for further investigation into the CS between tigecycline and colistin.

The past decade has witnessed the emergence and spread of carbapenem-resistant Enterobacter cloacae complex (CRECC), presenting a significant clinical challenge and urgently demanding new treatment strategies against antimicrobial resistance (AMR). This study focused on the impact of tigecycline on the susceptibility of CRECC isolates to colistin and the collateral sensitivity in CRECC. Under tigecycline pressure, the resistance of five clinically isolated CRECC strains to colistin was converted from resistance to sensitivity. These mutants exhibited significantly higher expression of acrA, acrB , and ramA genes, with mutations in the ramR gene. Overexpression of ramA in certain mutants did not alter ramR expression. No mutations were identified in lipid A synthesis genes; however, phoQ was consistently downregulated, and arnA expression varied among CRECC405-resistant mutants. Low-dose colistin and tigecycline combination therapy outperformed monotherapy in antimicrobial efficacy. Overall, collateral susceptibility to tigecycline was observed in CRECC isolates with colistin resistance. The overexpression of acrA, acrB, and ramA , due to ramR mutations, led to tigecycline resistance. Inconsistent expression levels of lipid A synthesis genes affected lipid A modification, which in turn upregulated arnA expression in CRECC405-resistant mutants. Increased sensitivity to colistin was associated with the downregulation of phoQ and arnA expression. IMPORTANCE: Antimicrobial resistance (AMR) is escalating faster than our ability to manage bacterial infections, with antibiotic-resistant bacteria emerging as a significant public health risk. Innovative strategies are urgently needed to curb AMR dissemination. Our research identified collateral sensitivity in Enterobacter cloacae complex following tigecycline (TGC) resistance, resulting in newfound sensitivity to colistin (COL), a drug to which it was once resistant. Synergistic tigecycline and colistin therapy significantly suppress bacterial growth, offering a promising approach to combat infections and curb AMR progression through the strategic pairing of antibiotics with complementary sensitivities.

Due to the complexity of identifying the Enterobacter cloacae complex (ECC) at the species level, little is known about the distribution of carbapenem-resistant ECC (CRECC). Plasmid-mediated mcr family genes are significant contributors to polymyxin resistance. The emergence of the mcr-9 gene has further complicated the landscape of polymyxin resistance in CRECC. Our study aimed to ascertain the prevalence of CRECC and the mcr-9 gene, and to elucidate the mechanisms underlying high-level resistance to polymyxin B (PB). In this study, we collected 212 non-replicating ECC strains, identifying 38 CRECC strains (17.9%, 38/212) and Enterobacter hormaechei (71.1%, 27/38) as the predominant endemic strains. Among these, 10 CRECC strains (36.3%, 10/38) were found to harbor the mcr-9 gene. Interestingly, the presence of mcr-9 did not significantly impact PB resistance or impose a fitness cost. While overexpression of mcr-9 can enhance PB resistance within a certain range and may incur fitness costs, it does not result in high-level PB resistance. The PB resistance of 17 CRECC strains was notably increased (from 16 to 128 mg/L), accompanied by mutations in the phoP/Q and mgrB genes. Notably, two novel insertion sequences, IS5D and IS1X2, were discovered within the mgrB gene. The inactivation of mgrB results in the loss of its negative regulatory effect on the two-component system. Protein structure predictions indicated that mutations in phoQ primarily affect the phosphatase (HAMP) and histidine kinase domains. This research significantly expands our comprehension of the complexities of PB resistance, highlighting the multifactorial nature of antibiotic resistance mechanisms.

Purpose Enterobacteriaceae carrying mcr-9, in particularly those also co-containing metallo-β-lactamase (MBL) and TEM type β-lactamase, present potential transmission risks and lack adequate clinical response methods, thereby posing a major threat to global public health. The aim of this study was to assess the antimicrobial efficacy of a combined ceftazidime/avibactam (CZA) and aztreonam (ATM) regimen against carbapenem-resistant Enterobacter cloacae complex (CRECC) co-producing mcr-9, MBL and TEM. Methods The in vitro antibacterial activity of CZA plus ATM was evaluated using a time-kill curve assay. Furthermore, the in vivo interaction between CZA plus ATM was confirmed using a Galleria mellonella (G. mellonella) infection model. Results All eight clinical strains of CRECC, co-carrying mcr-9, MBL and TEM, exhibited high resistance to CZA and ATM. In vitro time-kill curve analysis demonstrated that the combination therapy of CZA + ATM exerted significant bactericidal activity against mcr-9, MBL and TEM-co-producing Enterobacter cloacae complex (ECC) isolates with a 100% synergy rate observed in our study. Furthermore, in vivo survival assay using Galleria mellonella larvae infected with CRECC strains co-harboring mcr-9, MBL and TEM revealed that the CZA + ATM combination significantly improved the survival rate compared to the drug-treatment alone and untreated control groups. Conclusion To our knowledge, this study represents the first report on the in vitro and in vivo antibacterial activity of CZA plus ATM against CRECC isolates co-harboring mcr-9, MBL and TEM. Our findings suggest that the combination regimen of CZA + ATM provides a valuable reference for clinicians to address the increasingly complex antibiotic resistance situation observed in clinical microorganisms.

Isolates coproducing serine/metallo-carbapenems are a serious emerging public health threat, given their rapid dissemination and the limited number of treatment options. The purposes of this study were to evaluate the in vitro antibacterial activity of novel β-lactam-β-lactamase inhibitor combinations (BLBLIs) against carbapenem-resistant Klebsiella pneumoniae (CRKP) coproducing metallo-β-lactamase and serine-β-lactamase, and to explore their effects in combination with aztreonam, meropenem, or polymyxin in order to identify the best therapeutic options. Four CRKP isolates coproducing K. pneumoniae carbapenemase (KPC) and New Delhi metallo-β-lactamase (NDM) were selected, and a microdilution broth method was used to determine their susceptibility to antibiotics. Time-kill assay was used to detect the bactericidal effects of the combinations of antibiotics. The minimum inhibitory concentration (MIC) values for imipenem and meropenem in three isolates did not decrease after the addition of relebactam or varbobactam, but the addition of avibactam to aztreonam reduced the MIC by more than 64-fold. Time-kill assay demonstrated that imipenem-cilastatin/relebactam (ICR) alone exerted a bacteriostatic effect against three isolates (average reduction: 1.88 log₁₀ CFU/mL) and ICR combined with aztreonam exerted an additive effect. Aztreonam combined with meropenem/varbobactam (MEV) or ceftazidime/avibactam (CZA) showed synergistic effects, while the effect of aztreonam combined with CZA was inferior to that of MEV. Compared with the same concentration of aztreonam plus CZA combination, aztreonam/avibactam had a better bactericidal effect (24 h bacterial count reduction >3 log₁₀CFU/mL). These data indicate that the combination of ATM with several new BLBLIs exerts powerful bactericidal activity, which suggests that these double β-lactam combinations might provide potential alternative treatments for infections caused by pathogens coproducing-serine/metallo-carbapenems.

Carbapenem-resistant Enterobacterales (CRE) are some of the most important pathogens causing infections, which can be challenging to treat. We identified four bla _IMP -carrying CRE isolates and collected clinical data. The transferability and stability of the plasmid were verified by conjugation, successive passaging, and plasmid elimination assays. The IncC bla _IMP-4 -carrying pIMP4-ECL42 plasmid was successfully transferred into the recipient strain, and the high expression of traD may have facilitated the conjugation transfer of the plasmid. Interestingly, the plasmid showed strong stability in clinical isolates. Whole-genome sequencing was performed on all isolates. We assessed the sequence similarity of bla _IMP -harboring plasmid from our institution and compared it to plasmids for which sequence data are publicly available. We found that four bla _IMP -carrying CRE belonged to four different sequence types. The checkerboard technique and time-kill assays were used to investigate the best antimicrobial therapies for bla _IMP -carrying CRE. The time-kill assay showed that the imipenem of 1× minimum inhibitory concentration (MIC) alone had the bactericidal or bacteriostatic effect against IMP-producing strains at 4–12 h in vitro . Moreover, the combination of tigecycline (0.5/1/2 × MIC) and imipenem (0.5/1 × MIC) showed a bactericidal effect against the bla _IMP-26 -carrying CRECL60 strain. IMPORTANCE: Carbapenem-resistant Enterobacterales (CRE) are an urgent public health threat, and infections caused by these microorganisms are often associated with high mortality and limited treatment options. This study aimed to determine the clinical features, molecular characteristics, and plasmid transmissible mechanisms of bla _IMP carriage as well as to provide a potential treatment option. Here, we demonstrated that conjugated transfer of the IncC bla _IMP-4 -carrying plasmid promotes plasmid stability, so inhibition of conjugated transfer and enhanced plasmid loss may be potential ways to suppress the persistence of this plasmid. The imipenem alone or tigecycline-imipenem combination showed a good bactericidal effect against IMP-producing strains. In particular, our study revealed that imipenem alone or tigecycline-imipenem combination may be a potential therapeutic option for patients who are infected with IMP-producing strains. Our study supports further trials of appropriate antibiotics to determine optimal treatment and emphasizes the importance of continued monitoring of IMP-producing strains in the future.

Background Rapid, cost-effective, simple, and reliable bacterial typing methods are essential in clinical infection control. This study aimed to evaluate the clinical applicability and its consistency with conventional typing methods of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) typing function. Methods Here, we collected 126 carbapenem-resistant Klebsiella pneumoniae (CRKP) strains and 24 Enterobacter cloacae complex (ECC) strains, grouped them according to their strain characteristics, and clustered them using different methods, including MALDI-TOF MS, multi-locus sequence typing (MLST), core genome multi-locus sequence typing (cg-MLST), pulsed-field gel electrophoresis (PFGE). Results The results showed that MALDI-TOF MS was poorly matched with MLST and cg-MLST. MALDI-TOF MS was in relatively high agreement with PFGE for ECC, but for CRKP we concluded that the agreement was poor. We speculated that the reasons for this discrepant result might be the large difference in the number of the two strains and the fact that CRKP strains used to compare PFGE and MALDI-TOF MS were all from the ICU unit, which has a high degree of similarity. In addition, we found three possible outbreaks in our hospital after a small cluster analysis according to the time of strain isolation as well as the departments and wards of patients. Conclusion Therefore, although MALDI-TOF MS is less consistent with traditional methods in typing of these specific species, it may be one of the alternative tools for detecting outbreaks of strains carrying the carbapenemase gene to facilitate rapid clinical measures to prevent further spread of the outbreak.