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Development of a rapid diagnostic method for antimicrobial susceptibility testing of Enterobacterales using a drug susceptibility testing microfluidic device
Authors: Norihisa Nakayama
Abstract
Objective: The global spread of drug-resistant bacteria represents a critical public health challenge, underscoring the need for rapid and reliable antibiotic susceptibility testing. Recently developed drug susceptibility testing microfluidic (DSTM) devices enable rapid assessment by analyzing antibiotic-induced bacterial morphological changes. However, interpretation of these changes is complex and subjective, necessitating automation. In this study, we developed an image analysis–based interpretation system for DSTM devices and evaluated its performance and practical utility.
Methods : Using reference strains and 975 Enterobacterales isolates collected in Japan, we developed an algorithm that assigns susceptibility categories based on DSTM results. Eight antibiotics were evaluated: ampicillin (ABPC), cefazolin (CEZ), cefmetazole (CMZ), cefotaxime (CTX), cefepime (CFPM), meropenem (MEPM), levofloxacin (LVFX), and amikacin (AMK). Performance was assessed using 59 clinical isolates and 10 standard strains, with results compared with those obtained using the Clinical and Laboratory Standards Institute M07 broth microdilution method.
Results: Category agreement was highest for CEZ and LVFX (98.6%) and lowest for CMZ (82.6%). Major error rates were 0% for CFPM, LVFX, MEPM, and AMK, whereas ABPC and CTX showed the highest major error rate (7.2%). No very major errors were observed for CEZ; however, MEPM showed a relatively high very major error rate (13.0%). Although discrepancies occurred between algorithm-based and morphology-based interpretations for some antibiotics, morphological transitions generally corresponded to susceptibility profiles in most strains.
Conclusion: Image analysis–based automation reduced subjectivity associated with conventional visual evaluation and yielded highly reproducible results, enabling quickly determination of Enterobacterales antimicrobial susceptibility within 3 h.”
Other specific DSP article suggested by Editorial Board
Rapid species-level discrimination of pulmonary TB and NTM by metagenomic next-generation sequencing with concurrent respiratory microbiome profiling
Authors: Lu Li
Abstract
Introduction: Rapid discrimination between Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) remains clinically challenging, especially when conventional microbiological evidence is limited. Whether metagenomic next-generation sequencing (mNGS) can provide rapid species-level identification while simultaneously characterizing the respiratory microbiome remains to be systematically evaluated.
Methods: Bronchoalveolar lavage fluid from 74 retrospectively enrolled patients with clinically diagnosed pulmonary mycobacterial disease (62 TB, 12 NTM-pulmonary disease (NTM-PD)) was analyzed by mNGS. Conventional test results were extracted from medical records. A supplementary assessment excluding mNGS from diagnostic review was additionally performed to reduce potential incorporation bias. Microbial diversity and between-group differences in the respiratory microbiota were evaluated.
Results: In the clinically diagnosed cohort, mNGS was positive in 61/62 TB cases (98.4%) and 12/12 NTM-PD cases (100%). Mycobacterial cultures were negative in all tested patients in routine clinical practice. By comparison, AFB (8.82%, 3/34), T-SPOT.TB (71.43%, 10/14), and Xpert MTB/RIF (69.23%, 9/13) showed lower positivity among tested patients. In the supplementary assessment, 45/46 independently classified TB cases were mNGS-positive (97.8%). mNGS additionally detected non-mycobacterial pathogens in 62.16% (46/74) of patients, facilitating recognition of polymicrobial infection. Microbiome analysis revealed that the TB group showed relatively higher abundance of Streptococcus parasanguinis besides MTB, whereas NTM group was relatively enriched in opportunistic pathogens including Pseudomonas aeruginosa and Stenotrophomonas maltophilia.
Conclusion: In this retrospective real-world cohort, mNGS achieved rapid species-level discrimination of MTB and NTM with high positive detection rates, and simultaneously provided clinically relevant microbiome information, supporting its value as an adjunctive diagnostic tool for pulmonary mycobacterial infection.”
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Antibiotic stress triggers α-hemolysin-mediated hemolysis in E. coli. – An overlooked factor for hemolytic driving sepsis
Authors: Aman Singh
Abstract
This study demonstrates that exposure to sub-inhibitory concentrations of antibiotics can activate bacterial virulence in vitro. Under sub-minimum inhibitory concentration (sub-MIC) antibiotic stress, we investigated the regulation of α-hemolysin, a pore-forming exotoxin central to Escherichia coli-mediated cytotoxicity and sepsis. Hemolytic activity was assessed using goat erythrocytes following exposure of the hemolytic strain E. coli MTCC 729 to increasing sub-lethal concentrations of clinically relevant antibiotics, while the non-hemolytic strain E. coli MTCC 723 was included throughout as a biological negative control to validate assay specificity. Extracellular α-hemolysin levels were quantified by the Lowry protein assay, and toxin-induced lytic progression was examined using microscopic imaging and blood agar assays. Among the antibiotics tested, gentamycin (aminoglycoside; 92.15 % hemolysis at 30 min, 260 mg/L extracellular protein) elicited the strongest hemolytic response, significantly exceeding untreated controls (3.14 %), ciprofloxacin (fluoroquinolone; 74.11 %), tetracycline (broad-spectrum; 62.20 %), and ampicillin (β-lactam; 48.21 %) (p < 0.05). The absence of hemolysis in E. coli MTCC 723 cultures, together with Triton X-100 (96 %) and PBS (0 %) controls, confirmed the specificity of α-hemolysin-mediated cytolysis. Morphometric and time-lapse analyses further validated non-osmotic, pore-forming α-hemolysin activity, revealing a progressive cytolytic cascade characterized by erythrocyte crenation, membrane deformation, and terminal rupture. Collectively, these findings highlight the paradoxical nature of antimicrobial therapy, demonstrating that sub-therapeutic antibiotic exposure can potentiate toxin-mediated cytopathogenicity without inhibiting bacterial growth.
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Implementation of molecular screening for a more efficient Shiga toxin-producing Escherichia coli testing workflow
Authors: Quinn Patterson
Abstract
Culture-independent diagnostic tests (CIDT) have emerged as a preferred diagnostic approach for the rapid detection of etiologic agents of gastroenteritis. Although the relative ease and speed of CIDT support their use as a primary diagnostic test, there is still a need to isolate the organism from CIDT-positive specimens for disease surveillance. The objective of this study was to assess the performance of the testing algorithm used at the California Department of Public Health Foodborne & Waterborne Diseases Section (CDPH-FWDS) for Shiga toxin-producing Escherichia coli (STEC) recovery from CIDT-positive human stool specimens in transport medium, with particular focus on molecular screening of enrichment broths for a Shiga toxin gene (stx). When STEC was not isolated by culture of the submitted stool specimen, CDPH-FWDS subcultures from an enrichment broth started from the stool specimen. Enrichment broth can improve STEC recovery by allowing target bacteria to multiple while limiting the growth of normal fecal flora. The enrichment broths were screened for stx1 and stx2 using a lab developed real-time PCR assay. STEC was isolated from only 3% of the enrichment broths that were negative for stx1 and stx2 by molecular screening. This work demonstrates the utility of molecular screening of enrichment broths prior to culturing for bacterial isolation. Discontinuation of culture from stx molecular screen negative enrichment broths will result in decreased hands-on laboratory testing time and cost savings with minimal impact on STEC surveillance efforts.