Antimicrobial resistance (AMR) has emerged as a major threat to public health globally. Accurate and rapid detection of resistance to antimicrobial drugs, and subsequent appropriate antimicrobial treatment, combined with antimicrobial stewardship, are essential for controlling the emergence and spread of AMR. This article reviews common antimicrobial susceptibility testing (AST) methods and relevant issues concerning the advantages and disadvantages of each method. Although accurate, classic technologies used in clinical microbiology to profile antimicrobial susceptibility are time-consuming and relatively expensive. As a result, physicians often prescribe empirical antimicrobial therapies and broad-spectrum antibiotics. Although recently developed AST systems have shown advantages over traditional methods in terms of testing speed and the potential for providing a deeper insight into resistance mechanisms, extensive validation is required to translate these methodologies to clinical practice. With a continuous increase in antimicrobial resistance, additional efforts are needed to develop innovative, rapid, accurate, and portable diagnostic tools for AST. The wide implementation of novel devices would enable the identification of the optimal treatment approaches and the surveillance of antibiotic resistance in health, agriculture, and the environment, allowing monitoring and better tackling the emergence of AMR.

Background/Objectives: Antimicrobial resistance poses a major public health challenge. The World Health Organization has identified 15 priority pathogens that require prompt development of new antibiotics. This review systematically evaluates the antibacterial resistance of the most significant bacterial pathogens, currently available treatment options, as well as complementary approaches for the management of infections caused by the most challenging multidrug-resistant (MDR) bacteria. For carbapenem-resistant Gram-negative bacteria, treatment options include combinations of beta-lactam antibiotics and beta-lactamase inhibitors, a novel siderophore cephalosporin, known as cefiderocol, as well as older antibiotics like polymixins and tigecycline. Treatment options for Gram-positive bacteria are vancomycin, daptomycin, linezolid, etc. Although the development of new antibiotics has stagnated, various agents with antibacterial properties are currently in clinical and preclinical trials. Non-antibiotic strategies encompass antibiotic potentiators, bacteriophage therapy, antivirulence therapeutics, antimicrobial peptides, antibacterial nanomaterials, host-directed therapy, vaccines, antibodies, plant-based products, repurposed drugs, as well as their combinations, including those used alongside antibiotics. Significant challenges exist in developing new antimicrobials, particularly related to scientific and technical issues, along with policy and economic factors. Currently, most of the alternative options are not part of routine treatment protocols. Conclusions and Future Directions: There is an urgent need to expedite the development of new strategies for treating infections caused by MDR bacteria. This requires a multidisciplinary approach that involves collaboration across research, healthcare, and regulatory bodies. Suggested approaches are crucial for addressing this challenge and should be backed by rational antibiotic use, enhanced infection control practices, and improved surveillance systems for emerging pathogens.

Neisseria gonorrhoeae (N. gonorrhoeae) is the etiological agent of the second most common sexually transmitted disease in the world, gonorrhoea. Currently recommended and last available first-line therapy is extended-spectrum cephalosporins most often combined with azitromycin. However, misuse of antibiotics and the abilities of N. gonorrhoeae to acquire new genetic and plasmid-borne resistance determinants has gradually led to the situation where this bacterium has become resistant to all major classes of antibiotics. Together with a generally slow update of treatment guidelines globally, as well as with the high capacity of gonococci to develop and retain AMR, this may lead to the global worsening of gonococcal AMR. Since effective vaccines are unavailable, the management of gonorrhoea relies mostly on prevention and accurate diagnosis, together with antimicrobial treatment. The study overviews the latest results of mostly WHO-initiated studies, primarily focusing on the data regarding the molecular basis of the resistance to the current and novel most promising antibacterial agents, which could serve to establish or reinforce the continual, quality-assured and comparable AMR surveillance, including systematic monitoring and treatment with the use of molecular AMR prediction methods.

Infectious diseases remain a major global health concern, with a growing burden of antimicrobial resistance and consequent higher mortality in the human population. Accurate bacterial identification is fundamental across clinical, veterinary, agricultural, and research settings, supporting effective diagnosis, antimicrobial stewardship, infection control, food safety, and environmental monitoring; however, conventional approaches are limited by time constraints, reduced sensitivity, and challenges in detecting fastidious or uncultivable organisms. This review provides a comprehensive overview of classical and advanced methods, including microscopy, culture, biochemical testing, immunological and serological assays, proteomic and spectroscopy-based techniques, and molecular approaches, such as polymerase chain reaction (PCR), digital PCR, DNA hybridization, 16S rRNA gene sequencing, whole-genome sequencing, and metagenomics. The integration of artificial intelligence has further enhanced analytical performance. Nevertheless, harmonization of bioinformatics frameworks remains essential, as variability in algorithm-defined cut-off values limits standardized implementation of whole-genome sequencing in routine laboratories. Emerging technologies, including CRISPR-based diagnostics and phage- and nanomaterial-based detection systems, offer promising alternatives. Overall, the integration of these approaches is expected to improve the accuracy, speed, and applicability of bacterial identification across diverse settings; however, these advances should be implemented cautiously, with standardization remaining a key priority alongside technological modernization.

Background: In Serbia, mandatory PCV immunization was introduced in 2018, following a sequential PCV10/PCV13/PCV10 schedule. This study aimed to assess changes in pneumococcal serotype distribution, vaccine serotype coverage, antimicrobial resistance (AMR), and clonal dissemination of pediatric invasive and non-invasive isolates before and after PCV introduction. Methods: A total of 626 clinical pneumococcal isolates from children were analyzed, including 251 invasive (IPD) and 375 non-invasive (NIPD) isolates. Serotyping was performed using PCR and Quellung reactions, antimicrobial susceptibility testing followed EUCAST guidelines, and invasive isolates were further characterized by multilocus sequence typing (MLST). Serotype invasiveness ratios were evaluated in pre- (2010–2017) and post-PCV (2018–2024) periods. Results: Following PCV implementation, serotype coverage declined across all vaccines, most markedly for PCV10 (IPD: 67.7–35.6%; NIPD: 62.3–17.4%), with secondary reductions in PCV13, PCV15, and PCV20 coverage largely driven by the loss of PCV10 serotypes. The proportion of non-PCV20 serotypes rose from 7.2 to 22.9% in IPD and from 12.3 to 41.6% in NIPD, with NVTs being significantly more frequent in NIPD post-PCV period. Antimicrobial non-susceptibility decreased after PCV introduction, including significant reductions in penicillin and macrolide resistance in both IPD and NIPD isolates. Resistance levels remained higher among non-invasive isolates compared with invasive isolates. Multidrug resistance declined from 39.8 to 25.4% in IPD and from 61.0 to 29.5% in NIPD. MLST analysis showed broad clonal diversity, with ST473 and ST8144 predominating pre-PCV and ST1377 and ST320 post-PCV, together with the emergence of non-vaccine lineages and serotype switch in ST320 from 19F to 19A. Conclusion: Mandatory PCV immunization in Serbia has led to substantial shifts in pneumococcal serotype distribution, resembling patterns observed in countries relying predominantly on PCV10. Also, a reduction in antimicrobial resistance and an increase in genetic diversity were observed, highlighting ongoing serotype replacement and the need for continued integrated surveillance.