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Epidemiology and Genomics of Antimicrobial Resistance of Difficult-to-Treat Pathogens

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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: closed (31 March 2026) | Viewed by 6736

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Dr. Simone Ambretti

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1. Microbiology Unit, IRCCS Azienda Ospedaliero-Universitaria di Bologna, 40138 Bologna, Italy
2. Microbiology, Department of Medical and Surgical Sciences, University of Bologna, 40138 Bologna, Italy
Interests: epidemiology of bacterial and fungal infections; antimicrobial resistance
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Dear Colleagues,

Antimicrobial resistance (AMR) poses a significant global health threat, particularly with difficult-to-treat pathogens resistant to multiple or all available antibiotics. A multidisciplinary approach combining surveillance, molecular studies, and public health interventions is essential to combat this growing challenge. Epidemiology and genomics play crucial roles in understanding the spread, evolution, and mechanisms of AMR in these pathogens. Epidemiological studies can help to track the incidence, prevalence, and transmission dynamics of multi-drug-resistant organisms (MDRos) across different countries. They provide insights into risk factors, reservoirs, and the impact of antimicrobial stewardship policies. Genomics, on the other hand, allows for a deeper understanding of the genetic determinants of resistance, including mutations, horizontal gene transfer, and mobile genetic elements such as plasmids and transposons. Whole-genome sequencing (WGS) and metagenomics have revolutionized AMR research, enabling the identification of resistance genes, the tracking of outbreaks, and the characterization of pathogen evolution. Integrating epidemiological data with genomic analyses enhances our ability to predict resistance trends, develop rapid diagnostic tools, and inform targeted interventions.

This Special Issue welcomes the submission of manuscripts that further explore the epidemiology and genomics of AMR in difficult-to-treat pathogens, focusing on key bacterial species and resistance mechanisms.

Dr. Simone Ambretti
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 resistance
difficult-to-treat pathogens
epidemiology
genomics
MRSA
VRE
ESBL
CRE
CRAB
MDR pseudomonas aeruginosa

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Published Papers (6 papers)

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Research

19 pages, 3743 KB

Open AccessArticle

Phylogenetic Groups, Virulence Factors, and Antimicrobial Susceptibility of Escherichia coli Associated with Urinary Tract Infections from a Metropolitan Area of Buenos Aires, Argentina

by Nora B. Molina, Ramón A. González Pasayo, Marisa A. López and Mónica D. Sparo

Antibiotics 2026, 15(4), 350; https://doi.org/10.3390/antibiotics15040350 - 29 Mar 2026

Viewed by 486

Abstract

Background: Uropathogenic Escherichia coli (UPEC) is the primary etiological agent of urinary tract infections (UTIs) worldwide. The emergence of strains combining high virulence with multidrug resistance (MDR) poses a significant challenge to public health. This study aimed to characterize the phylogenetic distribution, virulence profiles, and antimicrobial susceptibility of UPEC isolates recovered from patients in the metropolitan area of Buenos Aires (AMBA), Argentina. Methodology: Phylogenetic groups, the ST131 lineage, and virulence-associated genes were identified using PCR-based assays. Antimicrobial susceptibility testing (AST) was performed using automated methods and extended-spectrum beta-lactamase (ESBL) production was confirmed using the double-disk synergy test. Colistin (COL) resistance was evaluated by Colistin Drop Test and PCR screening for the mcr-1 (mobile colistin resistance gene 1). Biofilm formation was detected by the Tissue Culture Plate (TCP) method, whereas phenotypic virulence factors (VF) were assessed with Congo Red agar, hemagglutination, and hemolysis assays. Results: Phylogenetic groups B2 (43.8%) and D (26.7%), typically associated with extraintestinal infections, were the most frequent. The high-risk clone B2-ST131 was detected in 6.7% of isolates. Biofilm production was observed in 92.4% of the isolates, with curli fimbriae (87.6%) being the most frequently expressed VF. The highest resistance rates were observed for ampicillin (62.1%), ampicillin-sulbactam (39.8%), and trimethoprim-sulfamethoxazole (25.2%). Interestingly, 3.8% of isolates exhibited colistin resistance, despite the absence of the mcr-1 gene. Conclusions: This study highlights the detection of MDR-UPEC isolates that showed strong resistance to fluoroquinolones and were ESBL producers with high virulence in Argentina, justifying future research encompassing genomic and epidemiological monitoring of local UPEC, which is essential for managing infections and developing new therapeutic and preventive measures. Full article

(This article belongs to the Special Issue Epidemiology and Genomics of Antimicrobial Resistance of Difficult-to-Treat Pathogens)

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14 pages, 1748 KB

Open AccessArticle

Molecular Characterization of Colistin-Resistant Clinical Acinetobacter baumannii from Northern Greece: Phenotypic Colistin Susceptibility and lpx/pmrCAB Mutational Profiles

by Dimitrios Karakalpakidis, Michaela-Eftychia Tsitlakidou, Michalis Paraskeva, Maria Nikoleta Mavidi, Maria Marinou, Kassandra Procter, Apostolos Beloukas and Christine Kottaridi

Antibiotics 2026, 15(3), 318; https://doi.org/10.3390/antibiotics15030318 - 20 Mar 2026

Viewed by 676

Abstract

Background: Acinetobacter baumannii (A. baumannii) is a formidable nosocomial pathogen and is classified by the World Health Organization (WHO) as a critical-priority pathogen, owing to its rapid evolution into extensively drug-resistant (XDR) and pan-drug-resistant (PDR) strains. Colistin remains one of the last-resort therapeutic options, although resistance rates are increasing in endemic regions such as Greece. In this study, we investigated the molecular basis of colistin resistance and characterized the clonal backgrounds of clinical XDR/PDR A. baumannii isolates collected between January and June 2022 from two tertiary-care hospitals in Thessaloniki, Northern Greece. Methods: We analyzed forty non-duplicate XDR/PDR clinical isolates. Antimicrobial susceptibility was determined using the VITEK 2 system, broth microdilution, and gradient diffusion methods. The lipid A biosynthesis genes (lpxA, lpxC, lpxD) and the pmrCAB operon were amplified by PCR and sequenced for all isolates. A representative subset of strains (n = 10/40) underwent multilocus sequence typing (MLST) according to the Pasteur MLST scheme. Results: All isolates proved colistin-resistant (MIC ≥ 4 µg/mL), and 95% were classified as PDR. Sequence analysis revealed multiple nonsynonymous mutations in the pmrCAB operon, with the PmrB A226V substitution predominating and extensive amino-acid changes observed in PmrC. In contrast, lpx genes exhibited limited protein-level variation, limited to lineage-associated polymorphisms (LpxC N287D, LpxD E117K). A novel six-nucleotide insertion in pmrB was identified in one isolate. MLST demonstrated a predominance of ST2 (International Clone 2), with single representatives of ST115 (IC2) and ST1 (IC1). Conclusions: In this cohort from Northern Greece, chromosomal mutations in the pmrCAB operon, within a predominantly ST2/IC2 background, were strongly associated with colistin resistance. These findings underscore the urgent need for continued molecular surveillance and targeted infection-control measures to limit further spread of PDR A. baumannii. Full article

(This article belongs to the Special Issue Epidemiology and Genomics of Antimicrobial Resistance of Difficult-to-Treat Pathogens)

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26 pages, 8655 KB

Open AccessArticle

Trends, Seasonality, and the Impact of COVID-19 on Clinical Staphylococcus aureus and MRSA Isolates in Western Mexico (2016–2025): A Time-Series Analysis at a University Referral Hospital

by Jaime Briseno-Ramírez, Pedro Martínez-Ayala, Adolfo Gómez-Quiroz, Brenda Berenice Avila-Cardenas, Brian Rafael Rubio-Mora, Roberto Miguel Damian-Negrete, Ana María López-Yáñez, Leonardo García-Miranda, Carlos Roberto Álvarez-Alba and Judith Carolina De Arcos-Jiménez

Antibiotics 2026, 15(3), 242; https://doi.org/10.3390/antibiotics15030242 - 25 Feb 2026

Viewed by 537

Abstract

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) remains a major cause of both community-onset and hospital-acquired infections, yet longitudinal data from Latin American hospitals spanning the COVID-19 pandemic are scarce. We characterized temporal trends, seasonality, and the impact of the COVID-19 pandemic on MRSA prevalence and incidence density among clinical S. aureus isolates at a tertiary-care hospital in western Mexico over 9.5 years. Methods: We analyzed 6625 non-duplicate clinical S. aureus isolates (6609 with valid resistance data) from June 2016 to December 2025. Temporal trends were assessed using Mann–Kendall tests, Theil–Sen estimation, and binomial generalized linear models. Seasonality was evaluated through STL decomposition, generalized additive models, and Fourier analysis. An interrupted time series (ITS) model with GLS-AR(1) and Newey–West corrections compared three COVID-19 phases: pre-pandemic (2016–2020), high viral circulation (2020–2022), and post-peak stabilization (2022–2025). Exposure-adjusted incidence densities (per 1000 patient-days) were analyzed in parallel. Results: MRSA prevalence declined from 28.1% pre-pandemic to 14.0% post-peak (Mann–Kendall z = −9.03, p < 0.001; OR = 0.85 per year, 95% CI: 0.829–0.871). MRSA incidence density decreased by 50%, from 1.27 to 0.63 per 1000 patient-days, while aggregate S. aureus incidence density remained stable (z = −0.17, p = 0.868). The ITS joint Wald test confirmed a significant cumulative shift in MRSA trajectory post-pandemic (p = 0.019 counts; p = 0.012 incidence density), with a significant post-peak level drop (p = 0.008). S. aureus exhibited moderate seasonality peaking in May–July (GAM edf = 7.26, p < 0.001), whereas MRSA showed only marginal seasonal variation. Conclusions: MRSA declined markedly across the study period, with the steepest reduction following the Omicron peak. The decline persisted after adjustment for pandemic-related fluctuations in hospital volume, supporting periodic reassessment of empiric anti-MRSA prescribing policies in similar settings. Full article

(This article belongs to the Special Issue Epidemiology and Genomics of Antimicrobial Resistance of Difficult-to-Treat Pathogens)

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14 pages, 279 KB

Open AccessArticle

Significant Prevalence of Dual KPC/NDM Carbapenemase-Producing Klebsiella pneumoniae in an ICU Cohort in Thessaloniki (2023), Including an ST512 Isolate Co-Harboring bla_NDM-1 and bla_KPC-3

by Maria Chatzidimitriou, Apostolos Voulgaridis, Pandora Tsolakidou, Fani Chatzopoulou, Ioannis Chonianakis, Eleni Vagdatli, Melania Kachrimanidou and Timoleon-Achilleas Vyzantiadis

Antibiotics 2025, 14(10), 994; https://doi.org/10.3390/antibiotics14100994 - 4 Oct 2025

Cited by 1 | Viewed by 1572

Abstract

Background/Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) threatens Intensive Care Units (ICU), particularly in settings where serine (KPC) and metallo-β-lactamases (NDM) co-circulate. The aim of this study was to assess CRKP susceptibility especially to novel β-lactam/β-lactamase inhibitor combinations, characterize the genetic determinants of resistance, and contribute to the understanding of local epidemiology in the ICU of our hospital. Methods: We studied 32 non-duplicate CRKP isolates (30 ICU, 2 wards) collected at Hippokration General Hospital, Thessaloniki (May–Oct 2023). Bacterial identification and Antimicrobial susceptibility testing (AST) were performed by VITEK-2; Minimum inhibitory concentrations (MICs) for ceftazidime/avibactam (CAZ/AVI), meropenem/vaborbactam (MER/VAB), and imipenem/relebactam (IMI/REL) were determined by E-tests. Colistin MICs were performed by broth microdilution. Carbapenemases were screened phenotypically and by immunochromatography and confirmed by multiplex PCR. One bronchial isolate co-harboring bla_NDM and bla_KPC genes underwent WGS. Results: All isolates were carbapenem-resistant and showed extensive resistance to β-lactams and fluoroquinolones. By PCR, 8/32 (25%) carried bla_KPC alone, 8/32 (25.0%) bla_NDM alone, and 16/32 (50%) co-harbored bla_KPC and bla_NDM. KPC-only isolates were generally susceptible in vitro to CAZ/AVI, MER/VAB, and IMI/REL, whereas dual KPC-NDM producers were resistant to all three combinations. Tigecycline showed the highest retained activity; colistin remained active in a minority. WGS of one ST512 (CG258) isolate revealed co-harboring bla_NDM-1 and bla_KPC-3 with additional resistance determinants and plasmid replicons, consistent with high-risk spread. Conclusions: Half of CRKP isolates in this ICU-predominant series co-produced KPC and NDM, severely limiting β-lactam/β-lactamase inhibitor options. These data support routine screening for carbapenemases, strict infection prevention, antimicrobial stewardship, and access to agents active against MBLs. Full article

(This article belongs to the Special Issue Epidemiology and Genomics of Antimicrobial Resistance of Difficult-to-Treat Pathogens)

9 pages, 215 KB

Open AccessCommunication

Rapid Emergence of Cefiderocol Resistance Associated with Mutation of EnvZ Gene in a VIM-Producing ST307 Klebsiella pneumoniae Strain

by Simone Ambretti, Benedetta Secci, Raul Cetatean, Milo Gatti, Pierluigi Viale, Federico Pea and Claudio Foschi

Antibiotics 2025, 14(9), 893; https://doi.org/10.3390/antibiotics14090893 - 4 Sep 2025

Cited by 1 | Viewed by 1154

Abstract

Infections caused by carbapenem-resistant Enterobacterales (CRE), particularly those harboring metallo-β-lactamases (MBLs) such as VIM, constitute a significant public health threat due to the paucity of effective therapeutic options. Cefiderocol (CFD), a novel siderophore-conjugated cephalosporin, exhibits potent activity against CRE by exploiting bacterial iron uptake systems. Nevertheless, the emergence of CFD resistance has been recently documented. This study aimed to characterize the development of CFD resistance in a VIM-producing Klebsiella pneumoniae isolate during antimicrobial treatment. Antimicrobial susceptibility was assessed by broth microdilution using iron-depleted medium according to EUCAST guidelines. Whole-genome sequencing and comparative genomic analyses focused on mutations in genes related to iron transport and CFD resistance, using Illumina MiSeq. Initial isolates (RS, BA1) were susceptible to CFD (MIC 2 mg/L), whereas the isolate recovered after 9 days of CFD therapy (BA2) was resistant (MIC 8 mg/L). In conclusion, this study illustrates for the first time the rapid emergence of CFD resistance in a VIM-producing ST307 K. pneumoniae isolate linked to a missense variant in envZ gene, arising after a 9-day CFD treatment. Full article

(This article belongs to the Special Issue Epidemiology and Genomics of Antimicrobial Resistance of Difficult-to-Treat Pathogens)

13 pages, 292 KB

Open AccessArticle

Molecular Detection of Multiple Antimicrobial Resistance Genes in Helicobacter pylori-Positive Gastric Samples from Patients Undergoing Upper Gastrointestinal Endoscopy with Gastric Biopsy in Algarve, Portugal

by Francisco Cortez Nunes, Catarina Aguieiras, Mauro Calhindro, Ricardo Louro, Bruno Peixe, Patrícia Queirós, Pedro Castelo-Branco and Teresa Letra Mateus

Antibiotics 2025, 14(8), 780; https://doi.org/10.3390/antibiotics14080780 - 1 Aug 2025

Viewed by 1744

Abstract

Background/Objectives: Helicobacter pylori (H. pylori) is a common gastric pathogen linked to gastritis, gastroduodenal ulcers, and gastric cancer. Rising antimicrobial resistance (AMR) poses challenges for effective treatment and has prompted the WHO to classify H. pylori as a high-priority pathogen. This study aimed to detect the prevalence of AMR genes in H. pylori-positive gastric samples from patients in Algarve, Portugal, where regional data is scarce. Methods: Eighteen H. pylori-positive gastric biopsy samples from patients undergoing upper gastrointestinal endoscopy were analyzed. PCR and sequencing were used to identify genes associated with resistance to amoxicillin (Pbp1A), metronidazole (rdxA, frxA), tetracycline (16S rRNA mutation) and clarithromycin (23S rRNA). Sequence identity and homologies were verified using tBLASTx and the Comprehensive Antibiotic Resistance Database (CARD). Results: Out of the 18 H. pylori-positive samples, 16 (88.9%) contained at least one AMR gene. The most frequent genes were rdxA (83.3%) and frxA (66.7%) for metronidazole resistance, and the 16S rRNA mutation (66.7%) for tetracycline. Resistance to amoxicillin and clarithromycin was detected in 27.8% and 16.7% of cases, respectively. Most samples (72.2%) had multiple resistance genes. A significantly strong association was found between female sex and the presence of the rdxA gene (p = 0.043). Conclusions: The study reveals a high prevalence of H. pylori resistance genes in Algarve, particularly against metronidazole and tetracycline. These findings highlight the need for local surveillance and tailored treatment strategies. Further research with larger populations is warranted to assess regional resistance patterns and improve eradication efforts. Full article

(This article belongs to the Special Issue Epidemiology and Genomics of Antimicrobial Resistance of Difficult-to-Treat Pathogens)

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