Search Results (94)

Background: Following a significant decline during the 2020–2021 SARS-CoV-2 pandemic, Mycoplasma pneumoniae (MP) experienced a resurgence across Europe in 2023–2024. Although macrolide-resistant MP has increased globally, severe disease can occur even in the absence of resistance, which highlights the importance of rapid molecular characterization for clinical purposes. In this context, clinical severity is often improperly used as a surrogate marker of macrolide resistance, potentially driving unnecessary antibiotic escalation. Methods: We report a severe MP pneumonia occurring during the 2023–2024 resurgence and evaluate macrolide resistance through a rapid two-step workflow (Real Time-PCR screening for A2063G/A2064G followed by confirmatory 23S rRNA sequencing), to assess whether severity predicts resistance and to support antibiotic stewardship. Results: The patient developed acute hypoxic respiratory failure (PaO₂ 54.9 mmHg; P/F ratio 110), extensive centrilobular micronodules on chest CT imaging, significant systemic inflammation and elevated liver enzymes. Respiratory support was escalated from a Venturi mask to a high-flow nasal cannula and BiPAP. MP infection was confirmed by multiplex Real Time-PCR (RT-PCR) and supported by positive IgM/IgG serology. RT-PCR targeting A2063G/A2064G mutations revealed no resistance-associated variants, and Sanger sequencing of an 807 bp 23S rRNA fragment confirmed a wild-type genotype. Despite severe hypoxemic respiratory failure, no resistance-associated variants were detected, documenting a clear severity–genotype mismatch. Clinical and radiological improvement followed second-line antibiotic therapy. Conclusions: Severe MP pneumonia can occur despite the absence of macrolide resistance. During MP re-emergence, clinical severity should not be used to infer macrolide resistance. Integrating nucleic acid amplification test (NAAT) diagnosis with rapid genotyping/confirmatory 23S rRNA sequencing can prevent misclassification, reduce unwarranted broad-spectrum escalation, and strengthen antimicrobial stewardship decisions. Full article

Staphylococcus aureus is a high-priority pathogen causing skin and soft tissue infections (SSTIs). The frequent resistance to anti-staphylococcal agents exhibited by this underscores the need for accurate diagnostics to guide effective therapy. Therefore, this study aimed to compare phenotypic and genotypic resistance in S. aureus isolates from nasal carriers and SSTIs and to elucidate gene-silencing mechanisms. In total, 355 S. aureus isolates (256 isolated from carriers and 79 from SSTIs) were studied for their phenotypic and genotypic resistance to β-lactams, macrolides, tetracyclines, aminoglycosides, and mupirocin. The silenced mupA gene (low prevalence: 0.6%; 2/335), linked to mupirocin resistance, was sequenced, and expression was assessed via reverse transcription qualitative PCR (RT-qPCR) in all mupA-positive isolates. SSTI isolates showed significantly higher resistance to erythromycin, gentamicin, and mupirocin, along with a higher prevalence of multidrug-resistant strains and ermC and tetM genes. Sequencing revealed multiple mutations in silent mupA, including a critical frameshift (c.372 delA) in a poly(A) tract that brings about premature truncation. RT-qPCR indicated upregulation of silent mupA variants and high variability in functional strains, suggesting that frameshift alone prevents resistance. These findings highlight silent resistance genes as key targets for advancing S. aureus surveillance and for combating emerging threats. Full article

Mycoplasma pneumoniae is a significant pathogen responsible for community-acquired respiratory infections in children and adolescents, with the rising prevalence of macrolide-resistant M. pneumoniae (MRMP), particularly in Asia, presenting critical treatment challenges. Our previous study inferred that a macrolide efflux pump may contribute to macrolide resistance in M. pneumoniae in addition to the common point mutations in 23S rRNA gene. This study aimed to define the specific pump and confirm its role. Through comparative genomic analysis, we identified a candidate gene, MPN_080, encoding an ABC transporter permease, which was further characterized using phylogenetic analysis, AlphaFold-based structural modeling, and biochemical assays. Overexpression of MPN_080 from an erythromycin-resistant isolate in the erythromycin-sensitive M129 resulted in a significant increase in minimum inhibitory concentrations (MICs) from <0.125 µg/mL to 1 µg/mL, while similar overexpression of MPN_080 derived from M129 did not affect MICs. Notably, this resistance mechanism operates independently of M. pneumoniae virulence factors, as evidenced by unaltered colonization capacity in NCI-H292 cells and consistent immune response patterns across both strains. Our findings establish MPN_080 as a novel determinant of macrolide resistance functioning associated with enhanced ATPase activity. These insights into non-classical resistance mechanisms may guide future diagnostic and therapeutic strategies against MRMP. Full article

Rhodococcus equi is an opportunistic intracellular pathogen responsible for severe pneumonia in foals and has emerged as an important cause of infection in immunocompromised humans. The treatment of R. equi infections in foals relies mainly on the combination of macrolides and rifampin. However, the increasing incidence of multidrug-resistant (MDR) isolates has raised significant therapeutic challenges. The mechanisms underlying this resistance include mutations in target genes, activation of efflux pumps, and biofilm formation, which collectively compromise the efficacy of conventional antibiotics. Recently, growing concern over antibiotic failure has accelerated research into alternative and synergistic strategies to enhance antibacterial efficacy and reduce the development of resistance. Natural and synthetic compounds, as well as optimized antibiotic combinations, have shown promising synergistic effects by enhancing intracellular accumulation, disrupting redox homeostasis, or inhibiting efflux systems. Experimental models employing checkerboard and time-kill assays, as well as redox-sensitive biosensors, have demonstrated that certain antibiotic combinations can influence bacterial susceptibility to antibiotic exposure. Furthermore, integrating molecular tools provides valuable insight into bacterial responses to oxidative and antibiotic stress, paving the way for novel therapeutic designs. This review summarizes the current understanding of the molecular factors contributing to antimicrobial resistance in R. equi and assesses new therapeutic approaches aimed at overcoming these challenges. It highlights recent findings on strategies to improve treatment outcomes and manage antimicrobial resistance. Full article

Background/Objectives: The aim of this study was to clarify the antimicrobial susceptibility and distribution characteristics of Mycoplasma pneumoniae (M. pneumoniae, MP) collected from children in Beijing, China, from 2017 to 2025. Methods: A total of 197 MP isolates were analyzed. Mutations in macrolide-resistant loci of MP strains were detected via real-time fluorescent quantitative polymerase chain reactions. We used the broth microdilution method to determine the minimum inhibitory concentrations (MICs) of erythromycin, azithromycin, tetracycline, levofloxacin, and moxifloxacin against these isolates. The distribution characteristics of MIC values were further analyzed according to the isolates’ collection year, epidemic phase (low epidemic phase, epidemic initiation phase, ultra-low epidemic phase, outbreak phase, and epidemic recovery phase), and the corresponding patient age group (<3 years, 3–6 years, and ≥6 years). Results: All 197 isolates were found to be resistant to erythromycin and azithromycin, with a resistance rate of 100%. In contrast, the strains remained susceptible to tetracycline, levofloxacin and moxifloxacin. The highest resistance rate was 100% for macrolides. The MIC₉₀ values were 1024 μg/mL for erythromycin, 256 μg/mL for azithromycin, 0.5 μg/mL for tetracycline, 1 μg/mL for levofloxacin, and 0.125 μg/mL for moxifloxacin, respectively. Distinct differences in MIC distributions of erythromycin and azithromycin were observed across collection years, epidemic phases, and age groups. Conclusions: The resistance of MP to macrolides in children is closely associated with the epidemic intensity and age of the patient. Erythromycin is no longer suitable as an empirical therapy for MP infections during epidemic periods, whereas azithromycin can be cautiously administered in young children according to age stratification and MIC detection results. Meanwhile, it is imperative to strengthen the prevention and control of cluster MP infections during epidemic phases to reduce the transmission of drug-resistant MP strains. Full article

Mycoplasma genitalium is a significant sexually transmitted pathogen, and its clinical management is increasingly complicated by the global distribution of mutations associated with macrolide and fluoroquinolone resistance. To characterize the molecular resistance landscape in a routine diagnostic setting, we retrospectively analyzed residual clinical specimens collected during routine sexually transmitted infection testing between January and December 2024. Among 374,021 specimens screened, we included 4019 M. genitalium-positive samples containing sufficient residual material. Using multiplex polymerase chain reaction assays, we detected mutations associated with macrolide and fluoroquinolone resistance in the 23S rRNA and parC genes, respectively. Frequent substitutions included A2059G and A2058G in the 23S rRNA gene (1253 samples) and substitutions at positions 248 and 259 in the parC gene (1306 samples). Mutations in the predefined 23S rRNA and/or parC targets were identified in approximately 44% of the analyzed samples, with distinct patterns of mutation distribution and co-occurrence. Although phenotypic susceptibility and clinical outcomes were not assessed, this large-scale, assay-based analysis provides a comprehensive overview of resistance-associated mutation patterns in M. genitalium derived from routine diagnostics, supporting molecular surveillance for monitoring antimicrobial resistance trends. Full article

Background: Bordetella pertussis causes pertussis, a respiratory infection with whooping cough. Despite a high vaccine coverage, pertussis resurged post-COVID-19 pandemic. Meanwhile, isolates resistant to macrolides—the first-line therapy—have increased in several countries, including Japan. Culturing B. pertussis and detecting resistance are difficult; reports remain limited in Japan. Methods: From March to August 2025, we collected nasopharyngeal samples from children aged 0–15 years with suspected pertussis at six Japanese clinics. Pediatricians obtained swabs and tested them using gene-amplification kits (e.g., BioFire^® SpotFire^® in four clinics, LAMP Pertussis Detection^® in two clinics). B. pertussis was confirmed by PCR; isolates were sequenced to identify macrolide-resistant mutations. Results: Samples were taken from 54 children, the number of boys and girls was 34 and 20, and their median age was 12 years old. Among 54 B. pertussis isolates, 43/52 (82.7%) sequenced strains harbored the A2047G mutation associated with macrolide resistance. Resistance rates at each clinic varied from 40% to 96%. Conclusions: These findings indicate a post-pandemic rise in macrolide-resistant B. pertussis in Japan. Ongoing resistance surveillance is essential, and repurposing residual clinical samples after routine testing is useful given culture and detection challenges. Full article

Background/Objectives: Antimicrobial resistance (AMR) represents a major global health challenge, driving the need for rapid and accurate diagnostic tools. Novel molecular assays, including multiplex PCR and DNA microarray-based systems, have emerged to detect antimicrobial resistance genes (ARGs) alongside bacterial identification. Methods: In this study, we evaluated the performance of the HybriSpot12 PCR AUTO (HS12a) system and the MDR Direct Flow Chip (MDR-FC) Kit—an automatic microarray assay based on reverse hybridization—for the detection of ARGs directly from positive blood culture (PBC) samples. A total of 111 Gram-negative bacterial isolates (92 Enterobacterales, 14 Acinetobacter baumannii, and 6 Pseudomonas spp.), previously characterized by whole-genome sequencing (WGS), were each used to generate a PBC, which was then analyzed with the HS12a/MDR-FC assay. Results: We demonstrated perfect agreement for the detection of macrolide resistance genes across all bacterial species and high agreement for genes conferring resistance to sulfonamides and β-lactams. In contrast, aminoglycoside resistance genes showed only moderate agreement, with minor discrepancies observed in Klebsiella pneumoniae and Escherichia coli, largely attributable to specific SNP variations. Conclusions: The HS12a/MDR-FC assay includes 51 ARGs, though not all were represented in our isolate set, and some false negatives were observed. Despite these limitations, its broad coverage and rapid turnaround remain advantageous compared to other rapid assays with fewer targets. Future refinements should aim at broader gene coverage, inclusion of key mutations, and detection of emerging variants, making this approach a promising tool for rapid AMR surveillance and antimicrobial stewardship. Full article

Campylobacter spp. remain among the most common pathogens causing acute diarrhea worldwide. Campylobacter jejuni and Campylobacter coli are the main species that cause gastroenteritis. Campylobacteriosis is a food-borne disease, although this Gram-negative bacterium may be transmitted via water-borne outbreaks as well as direct contact with animals, emphasizing its zoonotic potential. Campylobacterisosis does not usually require hospitalization. Antimicrobials are warranted only for patients with severe disease, as well as patients who are at risk for severe disease, such as the elderly, pregnant women or immunocompromised patients. Nonetheless, the irrational use of antibiotics in human and veterinary medicine enhances antimicrobial resistance (AMR). Resistance of Campylobacter spp. to fluoroquinolones, macrolides and tetracyclines is a significant concern to the scientific community. Point mutations, horizontal gene transfer and efflux pumps are the main mechanisms for the development and transmission of AMR in Campylobacter spp. Emerging evidence suggests that climate change may indirectly contribute to the spread of AMR in Campylobacter, particularly through its influence on bacterial ecology, transmission pathways and antibiotic use patterns. Higher temperatures and extreme weather events accelerate bacterial growth, amplify the transfer of AMR genes and magnify disease transmission, including drug-resistant infections. Horizontal gene transfer, especially in the context of biofilm formation, may further perplex the situation. Excessive farming and overuse of antibiotics as growth promoters in animals may also contribute to increased AMR rates. Climate change and AMR are interconnected and pose a significant threat to global public health. Multidisciplinary strategies mitigating both phenomena are crucial in order to contain the spread of Campylobacter-related AMR. The aim of this review is to describe the molecular mechanisms that result in AMR of Campylobacter spp. and underscore the association between climate change and Campylobacteriosis. Novel methods to mitigate Campylobacter-related AMR will also be discussed. Full article

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 bla_OXA-29, lpeAB or aph(9)-Ia gene. Results: AMR screening detected lpeAB, two allelic β-lactamase variants (bla_OXA-29 and bla_LoxA) 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 bla_OXA-29 and aph (9)-Ia. Full article

Mycoplasma pneumoniae (MP), an important pathogen that causes pneumonia among children and young adults, caused an epidemic every four years in Japan until 2016, with the next epidemic occurring eight years later in 2024. This study compared the prevalence of MP infections among Japanese children in 2024 to previous years using real-time polymerase chain reaction (PCR) and the p1 genotype determined using the PCR restriction fragment length polymorphism typing method from nasopharyngeal swab specimens. Of the 133 total isolates collected in 2024, 54.1% were macrolide-resistant MP (MRMP), with 98.0% of those containing an A2063G mutation in the 23S rRNA gene associated with macrolide resistance. This annual rate of MRMP and incidence of the A2063G mutation was similar to those in 2016. However, the dominant p1 genotype among isolates in 2024 was type 1 (93.4%), whereas type 2 was dominant in the previous epidemic. Thus, although the rate of MRMP in 2024 was similar to that in the previous epidemic year, the distribution of p1 genotypes was different. Further, the rate of MRMP was lower than neighboring Asian countries, including China and Korea, but was higher than in European countries. Therefore, it is important to continue monitoring MP infections in Japan. Full article

A post-COVID surge of Bordetella pertussis was observed globally. China has reported a high level of macrolide-resistant Bordetella pertussis (MRBP) in recent years; however, the epidemiology of MRBP in Hong Kong remains unknown. We retrieved archived B. pertussis isolates from respiratory samples collected at five regional public hospitals in Hong Kong between 2015 and 2024 and tested their minimum inhibitory concentration (MIC) for macrolides and other non-macrolide antibiotics using the Etest method. All isolates were also subjected to whole genome sequencing for genotypic resistance, Multi-locus Antigen Sequence Typing (MLST) and Multi-locus Variable Number of Tandem Repeat Analysis (MLVA) typing. Twenty-nine isolates of B. pertussis were included in the study. All isolates demonstrating phenotypic macrolide resistance harbored the A2047G mutation while showing low MIC to trimethoprim-sulfamethoxazole, doxycycline, levofloxacin, piperacillin-tazobactam and meropenem. In 2023 and 2024, 100% were MRBP and all belonged to the MT28 clone with the ptxP3 antigenic type. The MRBP isolates in Hong Kong were phylogenetically related to those from mainland China during the same period. There was no obvious correlation between macrolide resistance and clinical presentation, laboratory findings, management and outcome. Phylogenetic analysis suggests that MRBP isolates in Hong Kong and mainland China are closely related. Full article

The southern pudu (Pudu puda) faces significant threats from anthropogenic activities and infectious diseases. Using whole-genome sequencing (WGS) and forensic microbiology research, we describe a triple bacterial co-infection in a southern pudu impacted by wildfire disasters. The deer presented infected burn wounds on the extremities and dog bite wounds in the lumbosacral region, from which a multidrug-resistant CTX-M-1-producing Escherichia coli sequence type (ST) ST224 and a Klebsiella oxytoca ST145 were isolated, respectively. The patient died 13 days after admission in a wildlife rehabilitation center. During the necropsy, a sample from intracardiac blood was collected, and WGS analyses confirmed systemic dissemination of an E. coli ST224 clone. The broad virulome (adhesins, invasins, toxins, and immune evasion genes) and resistome against beta-lactams (bla_CTX-M-1), aminoglycosides [aac(3)-IId, aph(3′)-Ia, aph(3″)-Ib, aph(6)-Id], macrolides [mph(A)], sulfonamides (sul2), trimethoprim (dfrA17), and fluoroquinolones (gyrA and parC mutations) of E. coli ST224 contributed to the treatment failure and death of the wild animal. Additionally, an oval nodule was identified in the abdominal cavity caused by Acinetobacter baumannii ST1365, the first WGS-confirmed report in wildlife. This study highlights the value of applying forensic microbiology and WGS to investigate and understand One Health pathogens threatening wildlife impacted by natural and anthropogenic disasters. Full article

Campylobacter jejuni and Campylobacter coli are the two main campylobacter species that cause foodborne campylobacteriosis. Recent studies have reported that Campylobacter spp. are prone to developing resistance to antibiotics commonly used for their treatment, with many C. coli strains identified as multidrug-resistant. This study presents the results of the whole-genome sequencing analysis of the multidrug-resistant C. coli strain BCT3 isolated in Greece from a stool specimen of a pediatric patient presenting with diarrhea. The strain was isolated using selective culture media and, based on antimicrobial susceptibility tests, was found to be resistant to ciprofloxacin, tetracycline, erythromycin, azithromycin, clarithromycin, and doxycycline. To further characterize it, we performed whole-genome sequencing, which identified strain BCT3 as C. coli. Moreover, multilocus sequence typing assigned the BCT3 to the sequence type (ST) 872, belonging to clonal complex ST-828. The presence of multiple virulence genes revealed its pathogenic potential. The detection of antimicrobial resistance genes and mutated alleles was indicative of its resistance to fluoroquinolones, macrolides, and tetracyclines, supporting the observed phenotype. To our knowledge, this is the first reported clinical case of such a multidrug-resistant C. coli strain in Greece. Full article

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide which usually presents as mild, and self-limiting disease in immunocompetent individuals. However, in immunocompromised patients, such as those with common variable immunodeficiency, C. jejuni can cause severe recurrent infections requiring antibiotic treatment. Our study reports a case of a 37-year-old male patient with CVID, who had multiple episodes of C. jejuni intestinal infections over a 3.5-year period. A total of 27 stool samples were collected and analyzed between December 2020 and July 2024 during acute febrile diarrheal episodes, with C. jejuni isolated in 15 samples. Antimicrobial susceptibility testing (AST) during the course of the disease revealed three different antimicrobial resistance profiles including multi-drug-resistant phenotype. Whole genome sequencing was performed on three representative isolates, all identified as MLST type 367, ST-257 complex, with minimal genetic divergence, indicating a clonal origin. Genes and point mutations conferring resistance to macrolides, fluoroquinolones, beta-lactams, and tetracycline were identified in different C. jejuni isolates, along with key virulence factors linked to adherence, invasion, motility, and immune evasion. The genetic analysis of macrolide phenotypic resistance revealed different resistance mechanisms. Genotypic and phenotypic analyses of the same C. jejuni clone from single patient, and identified multidrug resistance pattern, present the first documented case of in vivo resistance development of C. jejuni in Croatia. This case highlights the role of prolonged antibiotic pressure in driving resistance evolution and underscores the need for careful antimicrobial stewardship and genomic monitoring in immunocompromised patients. Further research is needed to correlate phenotypic resistance with genetic determinants in Campylobacter spp. Full article