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Antibiotics
Special Issues
Advancing and Standardising Antimicrobial Susceptibility and Resistance Detection Methodology
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Advancing and Standardising Antimicrobial Susceptibility and Resistance Detection Methodology

A special issue of Antibiotics (ISSN 2079-6382). This special issue belongs to the section "Mechanism and Evolution of Antibiotic Resistance".

Deadline for manuscript submissions: 31 March 2026 | Viewed by 6640

Special Issue Editor

Prof. Dr. Owen B. Spiller

E-Mail Website
Guest Editor
1. Medical Microbiology, Division of Infection and Immunity, Cardiff University, Cardiff, UK
2. Bacteriology Reference Department, UK Health Security Agency, London, UK
Interests: antimicrobial resistance; neonatal sepsis; Ureaplasma; Mycoplasma; coagulase-negative staphylococci; legionella; group b streptococcus; Enterococcus spp.; Staphylococcus haemolyticus
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues, 

The rise of antimicrobial resistance (AMR) is one of the most pressing global health challenges of our time. The accurate and reliable detection of antimicrobial susceptibility and resistance is critical for guiding effective treatment strategies and curbing the spread of resistant pathogens. However, discrepancies in diagnostic methods, lack of standardisation, and limited access to advanced technologies in resource-limited settings hinder our ability to monitor and respond to AMR effectively.

This Special Issue aims to explore the latest advancements in antimicrobial susceptibility and resistance detection methodologies, focusing on the need for improved standardisation and consistency across clinical, environmental, and veterinary settings. Contributions are welcome to highlight innovative technologies, diagnostic platforms, and laboratory techniques that can enhance the accuracy, speed, and accessibility of AMR detection. Additionally, this Special Issue aims to address the challenges of validating these methodologies across diverse pathogen species, resistance mechanisms, and geographical regions.

We invite contributions that address, but are not limited to, the following topics:

  • Novel technologies for antimicrobial susceptibility testing (AST), such as next-generation sequencing, microfluidics, and biosensors.
  • Standardisation of AST protocols across clinical, veterinary, and environmental microbiology laboratories.
  • Comparative studies of traditional and emerging methods for AMR detection.
  • Point-of-care diagnostic tools for rapid and accurate AMR detection.
  • Quality assurance and control in AMR diagnostics.
  • The role of big data, machine learning, and artificial intelligence in improving resistance surveillance and prediction.
  • Global challenges in implementing standardised AMR detection in low-resource settings.

Through this Special Issue, we hope to promote the development of more robust and widely accessible detection methods, facilitating global efforts to fight AMR and improve patient outcomes. By standardising diagnostic approaches and embracing technological innovation, we can move closer to providing an effective response to one of the most urgent health threats of the 21st century.

Prof. Dr. Owen B. Spiller
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 250 words) can be sent to the Editorial Office for assessment.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Antibiotics is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2900 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • antimicrobial susceptibility testing
  • resistance screening
  • rapid diagnostics
  • clinical
  • veterinary
  • environmental
  • validation

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Further information on MDPI's Special Issue policies can be found here.

Published Papers (6 papers)

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Research

20 pages, 5934 KB  
Article
Antimicrobial Susceptibility Determination of Less Frequently Isolated Legionella Species by Broth and Agar Dilution
by Caitlin Farley, Amy Price, Max Sewell, Rachael Barton, Edward A. R. Portal, Ian Boostrom, Jessica Day, Baharak Afshar, Victoria J. Chalker and Owen B. Spiller
Antibiotics 2025, 14(11), 1165; https://doi.org/10.3390/antibiotics14111165 - 17 Nov 2025
Viewed by 471
Abstract
Background/Objectives: Infections caused by Legionella species are primarily associated with Legionella pneumophila, but non-pneumophila species are increasingly implicated in human disease. Despite this, antimicrobial susceptibility testing (AST) data for non-pneumophila species remain scarce, and standardised testing protocols or resistance [...] Read more.
Background/Objectives: Infections caused by Legionella species are primarily associated with Legionella pneumophila, but non-pneumophila species are increasingly implicated in human disease. Despite this, antimicrobial susceptibility testing (AST) data for non-pneumophila species remain scarce, and standardised testing protocols or resistance thresholds have not been established. This study aimed to address this gap by evaluating and comparing AST performance for non-pneumophila Legionella species relative to L. pneumophila using three methodologies. Methods: AST was conducted on 89 Legionella isolates using LASARUS agar dilution, buffered yeast extract broth microdilution (BYE-BMD), and BCYE-α agar dilution, against ampicillin, azithromycin, chloramphenicol, doxycycline, levofloxacin, and rifampicin. Growth performance and minimum inhibitory concentrations (MICs) were assessed after a 96 h incubation. Results: MIC profiles were obtained using LASARUS and BYE-BMD for 53.9% and 93.3% of isolates, respectively. While L. pneumophila reached sufficient turbidity in BYE-BMD after a 48 h incubation, non-pneumophila species required an extended incubation (72–96 h). Non-pneumophila species displayed broader MIC ranges against azithromycin (0.016–1 mg/L) and levofloxacin (0.016–0.25 mg/L), but a narrower rifampicin range (≤0.0005–0.032 mg/L) relative to L. pneumophila. L. longbeachae exhibited a higher MIC50 for rifampicin despite overlapping susceptibility ranges across all species (0.001–0.016 mg/L). Conclusions: This study demonstrates species-specific differences in Legionella susceptibility and highlights the limitations in extrapolating L. pneumophila-based AST data. Azithromycin MICs in non-pneumophila species exceeded those of L. pneumophila, raising clinical concern. While BYE-BMD was the most effective method for MIC determination, three species required BCYE-α due to poor growth. These findings support developing standardised, species-specific AST protocols and thresholds amid rising macrolide resistance and the increasing detection of non-pneumophila infections. Full article
(This article belongs to the Special Issue Advancing and Standardising Antimicrobial Susceptibility and Resistance Detection Methodology)
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16 pages, 3329 KB  
Article
Comparison of Phenotypic and Whole-Genome Sequencing-Derived Antimicrobial Resistance Profiles of Legionella pneumophila Isolated in England and Wales from 2020 to 2023
by Rediat Tewolde, Rebecca Thombre, Caitlin Farley, Sendurann Nadarajah, Ishrath Khan, Max Sewell, Owen B. Spiller and Baharak Afshar
Antibiotics 2025, 14(10), 1053; https://doi.org/10.3390/antibiotics14101053 - 21 Oct 2025
Viewed by 703
Abstract
Background: Antimicrobial resistance (AMR) in Legionella pneumophila is emerging as a concern, particularly with resistance to macrolides and fluoroquinolones. Although clinically significant resistance in Legionella pneumophila remains uncommon, systematic genomic surveillance using whole-genome sequencing (WGS) is needed to anticipate treatment failure as metagenomic [...] Read more.
Background: Antimicrobial resistance (AMR) in Legionella pneumophila is emerging as a concern, particularly with resistance to macrolides and fluoroquinolones. Although clinically significant resistance in Legionella pneumophila remains uncommon, systematic genomic surveillance using whole-genome sequencing (WGS) is needed to anticipate treatment failure as metagenomic diagnostics move toward routine use. Objectives: We assessed the UK Health Security Agency AMR pipeline for predicting resistance in L. pneumophila by analysing 522 L. pneumophila isolates from England and Wales (2020–2023) together with nine database sequences that carry confirmed 23S rRNA mutations conferring high-level azithromycin resistance. The objective of the present study was to examine the presence of antimicrobial resistance genes (ARGs) in L. pneumophila isolates and to determine whether they exhibited phenotypic resistance through minimum inhibitory concentration (MIC) testing. Methods: Serogroups (sgs) were determined using an in-house qPCR assay, and L. pneumophila non-sg1 isolates were serogrouped using the Dresden monoclonal antibody (mAb) typing method. Sequence types were determined using the standard sequence-based typing method by Sanger sequencing. WGS reads were screened against standard AMR databases to identify resistance genes and resistance-mediating mutations. Agar dilution measured MICs for azithromycin, erythromycin, ampicillin, levofloxacin, tetracycline and spectinomycin in isolates possessing the blaOXA-29, lpeAB or aph(9)-Ia gene. Results: AMR screening detected lpeAB, two allelic β-lactamase variants (blaOXA-29 and blaLoxA) and aph(9)-Ia in 165 of the 522 L. pneumophila isolates, while all high-azithromycin MIC reference sequences contained the expected 23S mutation. Only lpeAB was associated with a significant twofold elevation in macrolide MICs. Neither β-lactamase variant increased ampicillin MICs, and aph(9)-Ia carriage did not correlate with higher spectinomycin MICs. Conclusions: Advanced genomic analytics can now deliver timely therapeutic guidance, yet database-flagged genes may not translate into phenotypic resistance. Continuous pairing of curated mutation catalogues with confirmatory testing remains essential for distinguishing clinically actionable determinants such as 23S mutations and lpeAB from silent markers like blaOXA-29 and aph (9)-Ia. Full article
(This article belongs to the Special Issue Advancing and Standardising Antimicrobial Susceptibility and Resistance Detection Methodology)
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11 pages, 260 KB  
Article
Evaluation of the NG-Test CARBA 5 for Rapid Detection of Carbapenemases in Clinical Isolates of Klebsiella pneumoniae
by Bojan Rakonjac, Momčilo Djurić, Danijela Djurić-Petković, Jelena Dabić, Marko Simonović, Marija Milić and Aleksandra Arsović
Antibiotics 2025, 14(10), 989; https://doi.org/10.3390/antibiotics14100989 - 2 Oct 2025
Cited by 1 | Viewed by 1284
Abstract
Background: Carbapenem-resistant Klebsiella pneumoniae (CRKp) is a critical global health threat due to its multidrug resistance, primarily driven by carbapenemase production. Rapid and accurate detection of carbapenemases is essential for effective treatment and infection control. This study evaluates the validity of the NG-Test [...] Read more.
Background: Carbapenem-resistant Klebsiella pneumoniae (CRKp) is a critical global health threat due to its multidrug resistance, primarily driven by carbapenemase production. Rapid and accurate detection of carbapenemases is essential for effective treatment and infection control. This study evaluates the validity of the NG-Test CARBA 5, a rapid immunochromatographic assay, for detecting five major carbapenemases (KPC, NDM, VIM, IMP, OXA-48-like) in clinical CRKp isolates. Methods: Clinical isolates of CRKp were collected from various clinical specimens at the Military Medical Academy in Belgrade, Serbia, between January 2023 and October 2024. Detection of carbapenemases was performed using NG-Test CARBA 5, while PCR served as the reference method. Diagnostic performance was assessed by calculating sensitivity, specificity, and Cohen’s kappa coefficient. Results: Among 312 isolates, OXA-48-like was the most prevalent carbapenemase. NG-Test CARBA 5 showed high sensitivity (98.7%) and specificity (100%) overall, with excellent agreement for NDM (κ = 0.947), OXA-48-like (κ = 0.957), and KPC (κ = 0.978). However, it failed to detect VIM in five PCR-positive isolates, suggesting potential limitations. Conclusions: NG-Test CARBA 5 is a rapid and reliable tool for detecting major carbapenemases in CRKp, though its performance for VIM detection requires further investigation. This assay has the potential to improve clinical diagnostics and strengthen infection control in settings with high antimicrobial resistance. Full article
(This article belongs to the Special Issue Advancing and Standardising Antimicrobial Susceptibility and Resistance Detection Methodology)
11 pages, 1198 KB  
Article
Evaluation of a Novel Rapid Phenotypic Antimicrobial Susceptibility Testing System
by Yuan-Chao Xue, Filipe Cerqueira, Natalie Williams-Bouyer and Ping Ren
Antibiotics 2025, 14(10), 962; https://doi.org/10.3390/antibiotics14100962 - 25 Sep 2025
Viewed by 1109
Abstract
Background/Objectives: Phenotypic antimicrobial susceptibility testing (AST) is essential for guiding timely and effective antibiotic therapy. Rapid and accurate reporting of AST results enables earlier optimization of treatment and supports antimicrobial stewardship by minimizing unnecessary use of broad-spectrum antibiotics. This study aimed to evaluate [...] Read more.
Background/Objectives: Phenotypic antimicrobial susceptibility testing (AST) is essential for guiding timely and effective antibiotic therapy. Rapid and accurate reporting of AST results enables earlier optimization of treatment and supports antimicrobial stewardship by minimizing unnecessary use of broad-spectrum antibiotics. This study aimed to evaluate the performance of the Selux DX Next-Generation Phenotyping AST system in comparison with the standard-of-care MicroScan WalkAway Plus system and broth microdilution reference results. Methods: A total of 332 clinical isolates and 97 Antimicrobial Resistance (AR) Bank reference isolates were tested using the Selux DX and MicroScan systems. Performance was assessed by categorical agreement (CA), error rates [very major errors (VMEs), major errors (MEs), minor errors (mEs)], and turnaround time. Results: The Selux DX system demonstrated ≥90% CA for most drug–organism combinations, consistent with Clinical and Laboratory Standards Institute (CLSI) acceptance thresholds, although elevated error rates were noted for erythromycin, aztreonam, cefazolin, minocycline, and ampicillin/sulbactam. Across 5124 drug–bug combinations, 55 VMEs (1.1%), 42 MEs (0.8%), and 203 mEs (4.0%) were identified. The Selux DX system achieved a markedly shorter average turnaround time of 5.5 h compared with 16 h for the MicroScan system, though at the cost of a longer setup time. Conclusions: The Selux DX system provides rapid and reliable phenotypic AST results, supporting earlier clinical decision-making and antimicrobial stewardship. However, discrepancies with certain antimicrobial agents, particularly among highly resistant reference isolates, highlight the need for further validation in larger, multicenter studies. Full article
(This article belongs to the Special Issue Advancing and Standardising Antimicrobial Susceptibility and Resistance Detection Methodology)
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9 pages, 421 KB  
Article
Increase in Penicillin Non-Susceptibility in Group B Streptococci Alongside Rising Isolation Rates—Based on 24 Years of Clinical Data from a Single University Hospital
by Sunghwan Shin, Dong Hee Whang, Tae-Hyun Um, Chong Rae Cho and Jeonghyun Chang
Antibiotics 2025, 14(9), 928; https://doi.org/10.3390/antibiotics14090928 - 13 Sep 2025
Viewed by 953
Abstract
Background/Objectives: Streptococcus agalactiae (Group B Streptococci, GBS) is Gram-positive, beta-hemolytic coccus known to be transmitted by vertical transmission in neonates during birth with neonatal sepsis, pneumonia, and meningitis. In adults, particularly the elderly and those with diabetes mellitus, GBS can also cause [...] Read more.
Background/Objectives: Streptococcus agalactiae (Group B Streptococci, GBS) is Gram-positive, beta-hemolytic coccus known to be transmitted by vertical transmission in neonates during birth with neonatal sepsis, pneumonia, and meningitis. In adults, particularly the elderly and those with diabetes mellitus, GBS can also cause pneumonia and sepsis. Penicillin is the drug of choice, and GBS is generally susceptible to this antibiotic. This study investigates trends in GBS isolation rates and penicillin non-susceptibility over time at a university hospital. Methods: We retrospectively analyzed 24 years (2000–2023) of microbiological data from Ilsan Paik Hospital to investigate trends in GBS isolation and penicillin susceptibility. Isolates were identified and tested using the Vitek 2 system, following CLSI guidelines. WHONET 2023 was used for data aggregation and analysis. Trends were analyzed by dividing the study period into three intervals: Period 1 (2000–2009), Period 2 (2010–2019), and Period 3 (2020–2023). Antimicrobial susceptibility rates for total GBS and PCN-NS GBS (penicillin non-susceptible group B Streptococcus) were compared using chi-square tests. Results: Among 257,884 total isolates, 3003 (1.16%) were GBS, and 29 (0.97%) were PCN-NS. GBS and PCN-NS isolation rates increased significantly across the three periods (p = 0.0001 and p = 0.009, respectively). PCN-NS GBS showed reduced susceptibility to all tested antimicrobials, with no drug showing higher susceptibility compared to total GBS. Conclusions: This study demonstrates a statistically significant rise in both GBS isolation rate and penicillin non-susceptibility over time. Given the emergence of multidrug-resistant GBS strains, susceptibility testing and interdisciplinary collaboration between microbiologists and clinicians are critical to guiding effective antimicrobial therapy and preventing neonatal and adult GBS infections. Full article
(This article belongs to the Special Issue Advancing and Standardising Antimicrobial Susceptibility and Resistance Detection Methodology)
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17 pages, 2176 KB  
Article
Growth-Phase-Dependent Modulation of Quorum Sensing and Virulence Factors in Pseudomonas aeruginosa ATCC 27853 by Sub-MICs of Antibiotics
by Ahmed Noby Amer, Nancy Attia, Daniel Baecker, Rasha Emad Mansour and Ingy El-Soudany
Antibiotics 2025, 14(7), 731; https://doi.org/10.3390/antibiotics14070731 - 21 Jul 2025
Cited by 4 | Viewed by 1578
Abstract
Background: Antibiotics at sub-inhibitory concentrations can rewire bacterial regulatory networks, impacting virulence. Objective: The way that exposure to selected antibiotics (ciprofloxacin, amikacin, azithromycin, ceftazidime, and meropenem) below their minimum inhibitory concentration (sub-MIC) modulates the physiology of Pseudomonas aeruginosa is examined in [...] Read more.
Background: Antibiotics at sub-inhibitory concentrations can rewire bacterial regulatory networks, impacting virulence. Objective: The way that exposure to selected antibiotics (ciprofloxacin, amikacin, azithromycin, ceftazidime, and meropenem) below their minimum inhibitory concentration (sub-MIC) modulates the physiology of Pseudomonas aeruginosa is examined in this study using growth-phase-resolved analysis. Methods: Standard P. aeruginosa strain cultures were exposed to ¼ and ½ MIC to determine the growth kinetics under antibiotic stress. The study measured protease and pyocyanin production and the expression level of important quorum sensing and virulence genes (lasI/R, rhlI/R, pqsR/A, and phzA) at different growth phases. Results: Meropenem produced the most noticeable growth suppression at ½ MIC. Sub-MIC antibiotics did not completely stop growth, but caused distinct, dose-dependent changes. Azithromycin eliminated protease activity in all phases and had a biphasic effect on pyocyanin. Ciprofloxacin consistently inhibited both pyocyanin and protease in all phases. The effects of amikacin varied by phase and dose, while β-lactams markedly increased pyocyanin production during the log phase. In contrast to the plateau phase, when expression was often downregulated or unchanged, most quorum-sensing- and virulence-associated genes showed significant upregulation during the death phase under sub-MIC exposure. Conclusions: These findings indicate that sub-MIC antibiotics act as biochemical signal modulators, preserving stress-adapted sub-populations that, in late growth phases, activate quorum sensing and stress tolerance pathways. Full article
(This article belongs to the Special Issue Advancing and Standardising Antimicrobial Susceptibility and Resistance Detection Methodology)
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