Epithelial cell invasion is a key phenotype for many enteric pathogens, including Salmonella, Shigella, and Vibrio. An emerging invasive pathogen is adherent-invasive Escherichia coli (AIEC), which is implicated in the pathogenesis of inflammatory bowel disease (IBD). AIEC strains lack virulence factors commonly associated with other invasive pathogens, making genetic identification challenging. As a result, AIEC strains are classified phenotypically based on their ability to invade epithelial cells using a field-standard gentamicin protection assay. However, since AIEC strains are often isolated from IBD patients who receive various antibiotics as part of their treatment regimen, the rise of gentamicin-resistant isolates has made this assay less reliable for AIEC classification. To circumvent this issue, an alternative amikacin-based protection assay was developed to quantify and visualize AIEC cell invasion. This assay utilizes amikacin, a broad-spectrum semisynthetic aminoglycoside that effectively targets strains resistant to other aminoglycosides while remaining extracellular to eukaryotic host cells. Thus, extracellular bacteria are killed during infection while intracellular bacteria are "protected". This forms the basis of a quantitative assay to compare strain invasiveness, measured as the percent invasion score using colony-forming-units (CFU) recovered post-infection. This assay can also be used as a qualitative measurement of invasion by immunostaining infected epithelial cells and visualizing these cells through confocal microscopy. This method serves as a diagnostic tool to screen IBD patients for AIEC that can also be applied to other multidrug-resistant pathogens to assess cell invasion, a proxy for virulence and pathogenicity.

Canine pyoderma is a common bacterial skin infection predominantly caused by Staphylococcus species. This study, conducted from June to December 2024, analysed 71 PCR-confirmed cases of staphylococcal pyoderma to investigate epidemiological and host-associated risk factors and antimicrobial resistance (AMR) patterns. Bacterial isolates from PCR-positive samples were identified using MALDI-TOF MS and tested for antimicrobial susceptibility. Of the cases analysed, 63.38% involved monomicrobial infections, whereas 36.62% were accounted for polymicrobial infections. Staphylococcus pseudintermedius and Staphylococcus aureus were the predominant pathogens in both infection types, occurring alone or in combination with Escherichia coli, Pseudomonas aeruginosa or Klebsiella pneumoniae. Polymicrobial infections were significantly associated with recurrent disease, longer disease duration, tissue invasion and allergic skin manifestations. Both major Staphylococcus species exhibited high resistance to β-lactams and third-generation cephalosporins, with notable interspecies differences in resistance to ceftizoxime, trimethoprim-sulfonamide, amikacin, enrofloxacin and clindamycin. Although overall AMR did not differ significantly between major staphylococci species associated to monomicrobial and polymicrobial infections, however isolates from polymicrobial cases showed a consistent trend toward higher resistance across multiple antimicrobial classes. The findings emphasise the complexity of canine pyoderma characterised by bacterial communities dominated by Staphylococcus pseudintermedius and the increasingly relevant Staphylococcus aureus with potential zoonotic implications. Routine species-level identification, culture-guided therapy and continuous AMR surveillance are crucial for effective treatment and antimicrobial stewardship within a One Health framework.

We report a case of localized Mycobacterium abscessus complex pulmonary disease with phenotypic inducible macrolide resistance in a previously healthy woman in her 40s. Chest computed tomography (CT) showed a solitary thin-walled cavitary nodule with perilesional centrilobular nodules, confined to the right upper lobe. Bronchial lavage culture grew M. abscessus subsp. abscessus/bolletii. Clarithromycin susceptibility shifted from susceptible on Day 3 to resistant on Day 14. Combination antimicrobial therapy was initiated with imipenem/cilastatin, amikacin, azithromycin, clofazimine, and sitafloxacin. This led to rapid radiologic improvement, and video-assisted thoracoscopic wedge resection was performed 24 days after initiation of antimicrobial therapy. Culture and PCR of the resected specimen were negative for pathogens. The patient completed a 12-month course of antimicrobial therapy and remains recurrence-free at 30 months based on CT follow-up. The only notable side effect of chemotherapy was grade 2 skin hyperpigmentation, which was attributed to clofazimine.

Klebsiella pneumoniae (KPN) infections pose heightened risks in the geriatric population due to weakened immunity, prevalent comorbidities, potential exposure in long-term care settings, and increased likelihood of antibiotic resistance (ABR). The study focused on the prevalence and antibiotic resistance of KPN infections, the presence of ABR genes in KPN, and the genomic characterization of KPN obtained from geriatric patients in Chattogram. A total of 543 specimens were collected from four hospitals in Chattogram, along with demographic data from hospital records. Genomic DNA was extracted from multi-drug-resistant (MDR) KPN, and the presence of ABR genes, blaTEM-1, sul-1, aadB, blaNDM-1, blaSHV-11, and phoE was identified. To characterize the KPN genomes, two MDR KPN isolates were subjected to whole-genome sequencing (WGS), and the data were analyzed using bioinformatics tools to identify genomic determinants of ABR. KPN exhibited high resistance to ceftazidime (96%), cefuroxime (92%), and cefixime (83%), but sensitivity to colistin (79%) and amikacin (75%). MDR KPN was mostly detected in sputum (36%) and urine (27%) specimens, where the prevalence of ABR genes, blaTEM-1, sul1, aadB, blaNDM-1, and blaSHV-11 were 28.2%, 17%, 6.17%, 56%, and 48% of these strains, respectively. These genomes exhibited distinct profiles for sequence types, ST420 and ST277 in Kpn007 and Kpn016, respectively, and ABR genes (qnrS1, blaCTX-M-15, and blaSHV-27), virulence factors (ybt, iuc1, iro1), and contained both K (K20, K46) and O antigens (O1, O3b). MDR KPN in the geriatric population poses a serious health concern due to their increased vulnerability to infections and limited treatment options, requiring careful management.IMPORTANCEMultidrug resistance (MDR) and the hypervirulence nature of Klebsiella pneumoniae (KPN) in geriatric patients pose a critical health concern in nosocomial infections worldwide and result in high clinical complexity and mortality. The study investigated the factors for KPN infections and analyzed antimicrobial resistance profiles. More than 60% of Klebsiella pneumoniae isolates from geriatric patients were resistant to third- and fourth-generation cephalosporins, and most isolates carried blaNDM-1 and blaSHV-11 genes. Analyzing whole genomes of two KPNs, Kpn007 (ST277) was identified as a hypervirulent strain with aerobactin and yersinia siderophores, contributing to virulence, and Kpn016 (ST420) carried fluoroquinolone (qnrS1), ESBL (blaCTX-M-15 and blaSHV-27) resistance. Both genomes contained K antigens (K20 and K46) and O antigens (O1 and O3b).

Black Hairy Tongue(BHT)/Black tongue is usually an asymptomatic cosmetic nuisance, characterized by hyperkeratosis of the elongated filiform lingual papillae ('hairs'). Black hairy tongue is well known with various drugs. Due to this sign, many a times the drugs are withdrawn and replaced with other drugs. Imipenem and linezolid are the drugs used as antimicrobial drugs in a number of infections but has been repurposed in drug resistant tuberculosis. A case was referred to us who failed on longer MDR treatment for 14 months for further management. DST revealed resistance to 10 drugs and clinical resistance to additional 4 drugs, totalling 14 drugs amounting to extremely drug resistant tuberculosis(XXDR) coined for patients much beyond XDR TB. We put the patient on individualized regime which contained Imipenem, Amikacin, high dose moxifloxacin and Delaminid. Linezolid could not be used due to anaemia but introduced one month later. Patient developed BHT within a week, disappeared on withdrawing the Imipenem but reappeared on reintroduction. Imipenem causing BHT is reported in one case only in last 4 decades of its use as antimicrobial. We are reporting the second case. Patient also developed BHT and blackening of teeth with Linezolid. As Imipenem-cilastatin and Linezolid are important drugs especially in difficult to treat tuberculosis, instead of stopping them we continued with the above drugs despite persistence of black tongue and teeth under close supervision during the whole 18 months without any further complications and patient was cured. Black tongue and teeth also disappeared after stopping the treatment.

Nontuberculous mycobacteria (NTM) are environmental pathogens increasingly recognized as significant causes of pulmonary and extrapulmonary infections worldwide. This review provides a detailed overview of NTM, emphasizing their rising prevalence, evolving diagnostic frameworks, and complex treatment paradigms. The most commonly implicated species in human disease include Mycobacterium avium complex (MAC), M. abscessus, and M. kansasii. These organisms cause a spectrum of diseases, most notably NTM pulmonary disease (NTM-PD), especially in individuals with preexisting lung conditions, structural abnormalities, or immunocompromised states. The diagnosis of NTM-PD is often challenging due to clinical overlap with tuberculosis and other chronic respiratory diseases. The 2020 ATS/IDSA guidelines recommend a triad of clinical symptoms, radiographic findings (e.g., bronchiectasis or cavitary lesions), and microbiological confirmation (≥2 positive sputum cultures or one bronchoscopic lavage) for diagnosis. Accurate species identification and susceptibility testing are essential due to interspecies variation in drug responsiveness. Management of NTM-PD requires a multidisciplinary approach. Treatment regimens are guided by species type and drug susceptibility. Macrolide-based multidrug therapy is the cornerstone for MAC, while M. abscessus often requires prolonged intravenous therapy using amikacin, tigecycline, or imipenem, followed by oral agents. Novel therapies such as Omadacycline and Amikacin Liposome Inhalation Suspension (ALIS) offer promise for refractory cases. Non-pharmacological strategies, including airway clearance and nutritional support, are particularly important in bronchiectasis-associated disease. Management of NTM-PD requires a multidisciplinary approach. Treatment regimens are guided by species type and drug susceptibility. Macrolide-based multidrug therapy is the cornerstone for MAC, while M. abscessus often requires prolonged intravenous therapy using amikacin, tigecycline, or imipenem, followed by oral agents. The review also addresses briefly extrapulmonary manifestations of NTM infections, which may involve skin, lymph nodes, bone, and disseminated disease, especially in HIV-positive or immunosuppressed patients. Diagnostic vigilance is essential, as radiologic and microbiologic findings may be subtle or misleading. Coinfections with fungi or tuberculosis, and increasing reports of NTM following cosmetic or surgical procedures, further complicate clinical management. Given high relapse rates and complex drug regimens, long-term monitoring and individualized treatment plans are crucial. Future directions emphasize genotypic resistance testing, improved diagnostic criteria, and greater awareness among clinicians. As NTM incidence continues to rise worldwide, particularly in TB-endemic regions, timely diagnosis, tailored therapy, and expert consultation are vital for optimal outcomes. Clinicians must remain vigilant to address this growing public health concern effectively.

We investigated a case of postoperative osteomyelitis in a dog caused by methicillin-resistant Staphylococcus pseudintermedius (MRSP) at a veterinary teaching hospital in Brazil, and screened the cohabiting dog, five household members and the attending surgeon for MRSP and other methicillin-resistant staphylococci (MRS) during infection (2021) and two years later (2023). Nasal and skin swabs were cultured on oxacillin-supplemented media. Isolates were identified as MRSP by PCR and characterized by PFGE and whole-genome sequencing, including MLST, SCCmec typing, resistance gene profiling and core-genome SNP analysis. The index infection was caused by MRSP ST71-SCCmec II-III displaying multidrug resistance but amikacin susceptible; the dog was successfully treated with prolonged amikacin therapy combined with surgical revision. During infection, MRSP ST71-SCCmec II-III was recovered from the patient, four owners and the surgeon (6/8 colonized with MRSP), while the cohabiting dog carried methicillin-resistant S. epidermidis. Two years later, MRSP carriage was identified in the cohabiting dog and two owners (3/7), with co-existence of MRSP ST71-SCCmec II-III (cohabiting dog skin) and the emerging lineage ST1095-CC551-SCCmec V (cohabiting dog nares; two owners), while the patient and two other owners carried other MRS. Within each lineage, pairwise SNP distances were ≤ 8, consistent with recent transmission. Overall, this case illustrates long-term intra-household circulation and strain replacement of MRSP across dogs and humans, highlighting the importance of hygiene measures in households to reduce the risk of transmission.

Mycobacterium abscessus subsp. massiliense (Mabs) is an emerging nontuberculous mycobacterium associated with difficult-to-treat infections due to intrinsic antimicrobial resistance, intracellular persistence, biofilm formation, and limited responsiveness to currently available therapeutic regimens. In this context, adjuvant strategies targeting iron-dependent metabolic pathways and metal homeostasis may enhance the efficacy of conventional antimicrobials. This study investigated deferoxamine (DFO), a clinically approved iron chelator, as a potential adjuvant against Mabs using integrated in vitro and in silico approaches. Cytocompatibility was assessed using an MTT assay in RAW 264.7 macrophages and a hemolysis assay in human erythrocytes. Antimicrobial activity was evaluated through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays, while interactions with clarithromycin (CLA) and amikacin (AMK) were assessed using the checkerboard method. Effects on virulence-associated phenotypes were examined through biofilm formation assays and protein quantification in extracellular vesicle-enriched fractions. Intracellular activity and modulation of inflammatory mediator gene expression were investigated in Mabs-infected RAW 264.7 macrophages through colony-forming unit (CFU) recovery and reverse transcription quantitative polymerase chain reaction (qPCR). DFO exhibited low cytotoxicity and negligible hemolytic activity under the tested conditions. Direct antimicrobial testing revealed a predominantly bacteriostatic profile (MIC = 9.75 µg/mL; MBC > 10 mg/mL), whereas checkerboard analysis suggested a synergistic interaction with CLA (FICI = 0.047), which requires further confirmation by time-kill or CFU-based combination assays. Furthermore, DFO reduced biofilm biomass, decreased protein levels in vesicle-enriched fractions, lowered intracellular bacterial burden, and modulated cytokine gene expression in infected macrophages. Molecular docking, ADME/Tox, and PASS analyses generated exploratory hypotheses regarding potential molecular interactions and pharmacological properties. Overall, these findings support DFO as a promising experimental adjuvant candidate for further investigation against Mabs, particularly in combination with clarithromycin. However, confirmation of a putative iron-restriction-associated mechanism and its translational relevance will require validation in additional clinical isolates, iron-rescue experiments, mature biofilm models, and in vivo studies.

Objectives: To assess the feasibility and reproducibility of predicting antimicrobial resistance (AMR) in Escherichia coli from MALDI-TOF mass spectrometry data using a standardized, open-source machine learning (ML) workflow, we systematically compared four ML algorithms, evaluated the impact of culture conditions, extract storage, and spectral preprocessing on model performance, and validated results through nested cross-validation with statistical significance testing. Methods: A total of 282 clinical E. coli isolates were analyzed. Two MALDI-TOF MS datasets were generated from freshly cultured extracts (T1) and recultured isolates one year later (T3), yielding 4468 spectra. A third dataset from the T1 extracts stored at -20 °C for one year (T2) was evaluated for spectral stability but excluded from primary modeling likely due to storage-induced degradation. Protein spectra (m/z 2000-15,000) were preprocessed using an in-house developed MALDI-TOF preprocessing pipeline (MTPP) comprising variance stabilization, Savitzky-Golay smoothing, SNIP baseline correction, TIC normalization, LOWESS alignment, and MAD-based peak detection (SNR ≥ 3), yielding 121 m/z features. Four classifiers-Random Forest (RF), Logistic Regression, Support Vector Machine, and Gradient Boosting-were trained to predict resistance to 11 antibiotics using nested cross-validation: outer GroupShuffleSplit (5-fold, isolate-level) for evaluation and inner GroupKFold for recursive feature elimination (RFECV) and hyperparameter tuning (RandomizedSearchCV). Classification thresholds were optimized via the precision-recall curve. Model performance was assessed using AUROC, AUPRC, F1-score, Matthews Correlation Coefficient (MCC), and bootstrap 95% confidence intervals (1000 replicates). Pairwise model comparisons were tested with McNemar's chi-squared test. Results: Among the 12 antibiotics included in the analysis (meropenem excluded for absence of resistance), resistance prevalence ranged from 1.1% (colistin) to 59.9% (amoxicillin). Colistin was subsequently also excluded from ML modeling due to insufficient resistant isolates (n = 3), leaving 11 antibiotics for prediction. The best predictive performance was observed for ciprofloxacin (AUROC 0.76 [95% CI 0.74-0.77]; F1 0.54; MCC 0.38) and ceftazidime (AUROC 0.68 [0.65-0.71]; F1 0.36; MCC 0.29), using 13 and 37 RFECV-selected features, respectively. Amoxicillin achieved the highest F1-score (0.76), driven by high recall (0.98) but modest AUROC (0.58). No meaningful predictive signal was detected for amikacin, cefepime, or tigecycline (AUROC ≤ 0.57, F1 ≤ 0.17), attributable to extreme class imbalance, and no robust multi-peak resistance signature was detected in this dataset. McNemar's test confirmed that RF significantly outperformed Logistic Regression for all antibiotics (p < 0.01), while Gradient Boosting performed comparably to RF for ciprofloxacin (p = 0.17) and ceftazidime (p = 0.28). Frozen extracts (T2) produced lower spectral similarity and were excluded from model training; the aligned T1+3 dataset yielded the most stable performance across metrics. Conclusions: Machine learning analysis of MALDI-TOF spectra enables reproducible AMR prediction for selected antibiotics in E. coli, with ciprofloxacin and ceftazidime showing the strongest signal. Nested isolate-level cross-validation, multi-model comparison with statistical testing, and open-source code provide a transparent, reproducible foundation for integrating ML-assisted MALDI-TOF analysis into diagnostic AMR surveillance. Extract storage at -20 °C degrades spectral quality and should be avoided in ML training workflows.

This study was designed to achieve more effective treatment for patients and to encourage the development of new antibiotics, specifically targeting multidrug-resistant Escherichia coli. This bacterium is one of the primary causative agents of hospital-acquired infections (HAI). It is classified as a critical priority pathogen for the development of new antibiotics according to the World Health Organization (2024). In this study, 76 isolates from four bacterial genera were analyzed. E. coli was identified as the most prevalent infectious agent with 52% of the isolates, followed by Klebsiella pneumoniae (20%), Pseudomonas aeruginosa (12%), and Acinetobacter baumannii (16%). Internal Medicine was the hospital department with the highest frequency of E. coli infections. Sixty-five percent of the samples were derived from urine. This bacterium was more prevalent in females (57.5%) than in males (42.5%). The highest resistance rates were observed for Ampicillin and Ciprofloxacin, with 90% and 77.5% respectively, while the lowest resistance was found for the Carbapenems Ertapenem, Meropenem, and Aminoglycoside Amikacin, with 22.5%. Twenty-two point five percent of the E. coli isolates were classified as resistant, and 77.5% as multidrug-resistant. Sixty-two point five percent were extended-spectrum beta-lactamase (ESBL) producers. All of these isolates resisted Ampicillin, while 4% were resistant to Ertapenem, Meropenem, and Amikacin. E. coli was identified as the primary causative pathogen of HAI in the Hospital under study and demonstrated resistance to most currently prescribed antibiotics.

Mycobacterium abscessus is a rapidly growing nontuberculous mycobacterium that causes extrapulmonary infections, including surgical site infections. Mycobacterium abscessus subsp. abscessus (MAA) frequently develops macrolide resistance, resulting in difficult-to-treat infections. Recently, surgical site infections caused by MAAs after cosmetic surgeries have become a notable concern, particularly as the number of cosmetic surgery clinics in Japan continues to increase. Therefore, vigilance for MAA infections following cosmetic procedures is necessary. However, reports on the treatment of macrolide-resistant MAA are limited. We report a case of surgical site infection following cosmetic surgery caused by macrolide-resistant MAA. A 47-year-old woman developed subcutaneous erythema, induration, and fistula formation several months after a thread lift procedure. Exudate culture revealed MAA harboring a full-length erm41 gene associated with inducible macrolide resistance, and susceptibility testing confirmed macrolide resistance. Treatment was initiated with combination antimicrobial therapy, including imipenem/cilastatin, amikacin, and tigecycline for 4 weeks, followed by clofazimine and sitafloxacin for 11 months as maintenance therapy. Local debridement was performed; however, complete removal of the threaded material was not feasible because thread had dissolved. After 12 months of treatment, the fistula closed, and no recurrence occurred during follow-up. This case highlights the importance of considering MAA in surgical site infections following cosmetic surgery and suggests that individualized combination antimicrobial therapy based on susceptibility testing may lead to successful outcomes in macrolide-resistant extrapulmonary MAA.