Search Results (204)

Background/Objectives: Accurate determination of colistin (COL) in vitro activity against carbapenem-resistant Acinetobacter baumannii complex (CRAB) isolates remains challenging, as the reference broth microdilution (BMD) method is labor-intensive and not routinely implemented in most clinical laboratories. Semi-automated susceptibility methods for colistin in the clinical laboratory require validation. The present study evaluated the performance characteristics of the recently introduced Vitek^®2 card AST-N440 for COL antimicrobial susceptibility testing (AST) on CRAB isolates compared with a BMD-based reference method (ComASP Colistin). Methods: A total of 176 single-patient CRAB isolates from two distinct tertiary Greek hospitals between 2024 and 2025 were included. COL susceptibility testing was performed using Vitek^®2 AST-N440 and compared with BMD. Minimum inhibitory concentrations (MICs) were interpreted according to EUCAST breakpoints. Method performance was evaluated by calculating categorical (CA) and essential agreement (EA), sensitivity, specificity, positive and negative predictive values (PPV/NPV), and major (ME) and very major error rates (VME) according to ISO 20776-2. Results: Compared with BMD, AST-N440 showed a sensitivity of 89.6% and a specificity of 62.3%, with a PPV and NPV of 81.7% and 76.0%, respectively. The CA (80.1%) and the EA (46.0%) were below ISO acceptance criteria. The VME rate was 10.4%, and the ME rate 37.7%. Identical MIC values were observed in 25.0% of the isolates, while Vitek^®2 reported lower and higher MIC values than BMD in 46.6% and 28.4% of isolates, respectively. Conclusions: The Vitek^®2 AST-N440 card performed suboptimally for COL susceptibility testing in CRAB isolates. Further validation of automated systems for COL AST is needed. Full article

Background/Objectives: The Saker Falcon (Falco cherrug) is an endangered raptor species of ecological and conservation relevance. Despite its status, data regarding its microbiota and the prevalence of antimicrobial resistance (AMR) remain scarce, especially in Eastern Europe. This single-facility study aims to investigate the phenotypic and genotypic AMR profiles of Gram-negative bacteria isolated from captive Saker Falcons in Western Romania. Methods: Freshly voided fecal droppings were collected non-invasively from 40 clinically healthy Saker Falcons. Bacterial identification was performed using selective media and the VITEK^® 2 system. Antimicrobial susceptibility testing (AST) was conducted on a representative subset of 12 isolates. Selected resistance-associated genes were screened by conventional PCR. Results: Escherichia coli was the most prevalent 60% (n = 24/40), followed by Hafnia alvei 10% (n = 4/40) and Pseudomonas spp. 10% (n = 4/40). AST revealed phenotypic resistance among Enterobacteriaceae primarily to ampicillin 20% (n = 2/10), tetracycline 20% (n = 2/10), fluoroquinolones and sulfonamides 10% (n = 1/10), while susceptibility to imipenem 90% (n = 9/10) and gentamicin 90% (n = 9/10) remained high. The targeted resistance-associated genes were detected in selected phenotypically resistant isolates. PCR screening detected blaZ and ampC in 62.5% (n = 5/8) of tested isolates, blaOXA-61 in 37.5% (n = 3/8), blaOXA-51 in 25% (n = 2/8), tetK in 37.5% (n = 3/8), and gyrA in 12.5% (n = 1/8). The isolate used as the negative control, pansusceptible in AST, was confirmed negative for all targeted genes. Conclusions: This single-facility study provides baseline data on AMR traits in Gram-negative bacteria associated with Saker Falcons in Western Romania. Given the limited scale and isolate-based design of the study, the findings should be interpreted cautiously, but they support further investigation of wildlife-associated AMR within a One Health context. Full article

Antimicrobial resistance (AMR) is a silently escalating global crisis, presenting a specific challenge for the One Health approach. Landscapes can serve as reservoirs of AMR bacteria and genes, while synurban wildlife may act as vectors of bidirectional exchange. However, these species can also be utilised as sentinels of landscape AMR load. Herbivorous avian bioindicators, such as the Common Wood Pigeon (Columba palumbus), continuously sample the landscape during foraging and drinking, providing a homogenous spatial overview on the state of AMR. This study aimed to investigate the potential of this species for assessing the impact of landscape diversity on bacterial communities and their AMR patterns. Toward this objective, two spatial units of 4 km in diameter located at an upstream and a downstream section of a river, relative to a provincial town, were compared using 16 cloacal samples per site. Heterotrophic plate count techniques resulted in 60 isolates, of which 48 were identified, and 35 were tested for AMR using the VITEK 2 Compact system. The association between bacteriological findings and landscape diversity was analysed using Rényi diversity profiles. The Gram-positive/Gram-negative ratio was the only parameter that proved to be significantly different between the two study sites. The investigation detected six MDR isolates, with two methicillin-resistant phenotypes (Staphylococcus gallinarum, Mammaliicoccus lentus) and two carbapenem-resistant Pseudomonas fluorescens isolates. We established that the small sample size limits drawing definitive conclusions. However, exploring the link between landscape diversity and the cultivable faecal microbiota of herbivorous birds is a promising approach for more extensive research. Full article

Background/Objectives: Antimicrobial resistance (AMR) in companion animals is an emerging public health threat due to zoonotic potential and limited therapeutic options. Dogs with otitis externa may harbor multidrug-resistant (MDR) bacteria, including Escherichia coli (E. coli), Proteus mirabilis (P. mirabilis), and Enterobacter cloacae complex (E. cloacae complex), some producing extended-spectrum beta-lactamase (ESBL) or AmpC β-lactamases. This study aimed to assess the prevalence, AMR patterns, MDR occurrence, β-lactamase production, and co-infection profiles of these pathogens in canine otitis externa. Methods: Ear canal samples were collected from 592 dogs presenting clinical signs of otitis externa, with one sample per dog included in the analysis. Samples were collected from veterinary clinics in Timiș County, Romania, from 2022 to 2025. Samples were cultured on blood agar and MacConkey agar, followed by biochemical testing and MALDI-TOF mass spectrometry for bacterial identification. Antimicrobial susceptibility testing against 15 agents across six classes was performed using the VITEK^® 2 system. MDR and β-lactamase production (ESBL, AmpC) were determined according to CLSI 2018 veterinary guidelines. Co-isolation with bacterial and fungal species were recorded. Results: E. coli, P. mirabilis, and E. cloacae complex were isolated in 9.12%, 6.25%, and 1.2% of cases, respectively. E. coli exhibited the highest resistance to aminoglycosides (tobramycin 72.2%, gentamicin 61.1%) and full susceptibility to carbapenems. P. mirabilis showed the highest resistance to ampicillin (54%) and trimethoprim + sulfamethoxazole (46%), with complete susceptibility to carbapenems and fluoroquinolones. E. cloacae complex displayed universal resistance to cephalosporins but remained susceptible to non-cephalosporin β-lactams (piperacillin–tazobactam), carbapenems and aminoglycosides. MDR prevalence was 35.2% for E. coli, 18.9% for P. mirabilis, and 14.3% for the E. cloacae complex. ESBL production was detected in 13% of E. coli and 8.1% of P. mirabilis isolates, while all E. cloacae complex isolates were AmpC-positive. Co-isolations were common, primarily involving Staphylococcus pseudintermedius (S. pseudintermedius) and Malassezia pachydermatis (M. pachydermatis). Conclusions: MDR and β-lactamase-producing bacteria were identified in dogs with otitis externa, emphasizing the importance of routine antimicrobial susceptibility testing, targeted therapy based on local resistance profiles, and continuous AMR surveillance to prevent treatment failure and mitigate zoonotic risk. Full article

Background/Objectives: Data on antimicrobial resistance (AMR) in goat-derived E. coli within the Gulf Cooperation Council (GCC) region remain limited, and are largely restricted to studies conducted in Saudi Arabia and the UAE, with no published reports from Qatar. This study provides the first baseline characterization of AMR and extended-spectrum β-lactamase (ESBL) profiles of E. coli isolated from goats in Qatar. Methods: A total of 280 fecal samples were collected from goats across nine locations in Qatar (140 healthy and 140 diseased goats; 12 samples did not yield E. coli cultures). A selective agar medium was used to isolate E. coli, and the isolates were subsequently confirmed using the VITEK^® 2 Compact system. Antimicrobial susceptibility testing was performed to determine resistance profiles, and PCR assays were used to detect ESBL-associated genes. Results: 268 E. coli isolates were recovered from 280 samples. AMR analysis revealed a high prevalence of tetracycline resistance among E. coli isolates (53%), consistently observed across all nine sampling locations. Ampicillin resistance was also widespread. AMR was detected in isolates from both healthy and diseased goats; however, gentamicin resistance was found exclusively in the isolates from diseased animals. Overall, 44 isolates (16%) were classified as multidrug resistant (MDR), while nine isolates (3%) demonstrated ESBL production based on cefotaxime resistance. MDR and ESBL-producing E. coli were detected across all nine locations and in both healthy and diseased animals, with MDR strains occurring more frequently than ESBL producers. PCR analysis identified ESBL-associated genes, namely, bla_CTX-M in nine isolates and bla_TEM in three isolates. Conclusions: Goats in Qatar harbor multidrug-resistant and ESBL-producing E. coli, highlighting their role as AMR reservoirs within a One Health framework. The high resistance rates to commonly used antibiotics, particularly tetracycline and ampicillin, across health statuses and geographic locations suggest potential influences of local management practices and environmental factors. The detection of ESBL genes, notably bla_CTX-M and bla_TEM, underscores the need for prudent antimicrobial use and the implementation of integrated One Health surveillance programs to mitigate potential public health risks and to support national AMR surveillance and antimicrobial stewardship efforts across the region. Full article

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

(1) Background: Chronic rhinosinusitis (CRS) with recurrent symptoms despite therapy raises concern for underlying antimicrobial resistance. While inflammation is central to disease pathophysiology, increasing evidence suggests that resistant bacterial populations within the sinonasal niche may contribute to treatment failure. This study aimed to characterize the molecular resistance profiles of bacterial isolates from refractory CRS patients and evaluate genotype–phenotype concordance and clinical resistance burden. (2) Methods: Our observational study includes 99 bacterial isolates obtained by endoscopically guided nasal swabs from adult CRS patients with recurrent disease. Species identification and antimicrobial susceptibility testing were performed using the VITEK^®2 system. Resistance genes were detected using multiplex-PCR. Statistical analyses included Mann–Whitney U tests for genotype–phenotype associations, Kruskal–Wallis testing across MDR categories, Spearman correlation between gene burden and clinical risk, and concordance metrics. (3) Results: Recognized sinonasal pathogens accounted for 46.5% of isolates, predominantly Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa. β-lactamase genes (tem 25.3%, shv 9.1%, ctxM 8.1%) and macrolide resistance markers (ermB 20.2%) were most prevalent, while carbapenemase genes remained infrequent. Significant phenotype–genotype correlations were observed for mecA–oxacillin, sul1–TMP-SMX, KPC–meropenem, and tem–β-lactams (p < 0.01). Gene burden increased progressively across clinical risk categories (p < 0.001), with MDR/XDR isolates concentrated in patients with repeated antibiotic exposure. Molecular and phenotypic analyses demonstrated high concordance for selected gene–antibiotic pairs, supporting targeted molecular screening as an adjunct to culture-based diagnostics in refractory CRS. Full article

Widespread release of cyanide from industrial activities represents a significant environmental challenge due to its acute toxicity and adverse effects on biological systems. In response to this concern, this study focused on the production of rhodanese from Kocuria rhizophila under submerged fermentation conditions and the assessment of its relevance for cyanide detoxification applications. A soil-derived Gram-positive bacterium was isolated and identified as Kocuria rhizophila based on morphological traits, biochemical profile-based VITEK 2 analysis, and 16S rRNA gene sequencing. Preliminary screening confirmed rhodanese production with an activity of 0.968 RU/mL. Under cyanide-induced submerged fermentation, enzyme production followed a growth-associated pattern and reached maximal activity at 40 h under optimized conditions (35 °C, pH 8.0). Partial purification using sequential precipitation and chromatographic steps enhanced enzyme purity, and SDS–PAGE analysis of the final fraction revealed protein bands at approximately 40, 140, and 260 kDa. Biochemical characterization showed Km values of 33.9 mM for KCN and 19.7 mM for sodium thiosulfate, with a Vmax of ~5.6 µmol min⁻¹ mL⁻¹ for KCN and optimal activity at pH 7–8 and 35 °C. Functional assays demonstrated efficient cyanide detoxification, achieving >85% conversion of KCN, ~92% of NaCN, and 65–77% of Ca (CN)₂ within 60 min in vitro. Collectively, these findings demonstrate that Kocuria rhizophila represents a promising microbial source of rhodanese with efficient cyanide-detoxifying activity, highlighting its potential for biotechnological and environmental remediation applications. Full article

Aim: Infections and multidrug-resistant (MDR) pathogens are concerns in pediatric palliative care (PPC) units, where children with life-limiting conditions undergo invasive procedures and prolonged hospitalization. This study evaluated clinical characteristics, microbiological profiles, and factors associated with MDR infections among pediatric patients hospitalized in a PPC unit. Methods: This retrospective observational study included 66 children aged 1 month to 18 years who were admitted to the PPC unit of our hospital due to infection between June 2023 and January 2024. Demographic data, comorbidities, device use, infection sites, and microbiological results were reviewed. Bacterial identification and antimicrobial susceptibility testing were performed using the Vitek2 system and interpreted according to EUCAST. Results: The median age was 48 months (IQR 19–106); 63.6% were male. Lower respiratory tract infection was most common (68.2%), followed by sepsis (13.6%) and urinary tract infection (12.1%). Pseudomonas aeruginosa (36.4%) and Klebsiella pneumoniae (27.3%) predominated. MDR organisms represented 15.2% of isolates. MDR infections were significantly associated with percutaneous endoscopic gastrostomy or mechanical ventilation use (p = 0.033). Prolonged hospitalization and multiple comorbidities tended to increase the MDR risk but did not reach statistical significance. Conclusions: Gram-negative MDR infections constitute an important problem in PPC units. Frequent exposure to invasive devices and antibiotics increases susceptibility to resistant pathogens. Reinforcing infection prevention, optimizing antimicrobial stewardship, and monitoring device-related infections are essential to reduce morbidity and improve care quality in pediatric palliative care. Full article

Background: The Trichosporonaceae family includes genera such as Trichosporon, Apiotrichum, and Cutaneotrichosporon, which are components of the human microbiota but may cause infections under conditions such as immunosuppression, prolonged hospitalization, invasive procedures, and broad-spectrum antimicrobial use. Objectives: This study aimed to describe the clinical and epidemiological characteristics of hospitalized and outpatient individuals with positive cultures for Trichosporonaceae species in Juiz de Fora, Minas Gerais, Brazil, and to correlate these findings with antifungal susceptibility profiles. Methods: Clinical isolates collected between 2020 and 2023 were identified using the Vitek 2^® system, and antifungal susceptibility was assessed by disk diffusion. Clinical and epidemiological data were obtained from electronic health records. Results: Among 40 isolates, Trichosporon asahii predominated (92.5%). Most cases involved hospitalized individuals (83.8%), mainly from intensive care units (81.8%). Respiratory infections and acute renal failure were the most common reasons for admission. The mean hospital stay was 34.8 days, and overall mortality reached 51.6%. Most individuals were male (77.5%) and older than 61 years (57.5%). Urine was the most frequent specimen (52.5%), and invasive infections predominated (87.5%). Corticosteroid use and invasive devices were common, and prior antibiotic use occurred in most cases. Only 35.0% of patients received antifungal therapy, predominantly fluconazole. Conclusions: Trichosporonaceae infections, particularly those caused by T. asahii, are associated with critically ill patients and high mortality, highlighting the need for early diagnosis, appropriate therapy, and continuous surveillance. Full article

Background: Bovine mastitis is a leading cause of economic loss in dairy farming and is increasingly complicated by antimicrobial resistance (AMR), posing challenges to treatment and public health. Objectives: This study aimed to investigate the prevalence, bacterial etiology, and AMR patterns of mastitis pathogens in dairy herds from the Banat region of Romania. Materials and Methods: A retrospective analysis was conducted on 420 dairy cows from five localities. Mastitis diagnosis involved clinical examination, indirect tests (California Mastitis Test (CMT), R-Mastitest), and bacteriological culture. Antimicrobial susceptibility was assessed using the VITEK^® 2 system. Results: Out of 420 cows, 120 (28.6%) were diagnosed with mastitis. The predominant pathogens were Staphylococcus aureus (33.3%) and Streptococcus agalactiae (22.5%). Most infections were monomicrobial (70%) and affected a single under quarter (77.5%). Beta-lactam resistance was widespread among both Gram-positive and Gram-negative isolates, particularly against penicillin and ampicillin. Multidrug-resistant (MDR) strains were identified in 33.3% of all isolates, with 100% of Gram-negative isolates exhibiting MDR profiles. Conclusions: The high prevalence of S. aureus and S. agalactiae, along with widespread beta-lactam resistance and frequent MDR phenotypes, highlights the urgent need for routine AMR surveillance and targeted antimicrobial therapy in bovine mastitis control programs. Full article

Background/Objectives: Recent investigations point to red foxes (Vulpes vulpes) as a very potent sentinel species for monitoring the dissemination of antimicrobial bacteria in wildlife habitats. Methods: This study investigated antimicrobial resistance in red foxes from 16 hunting grounds (peri-urban and peri-rural) in western Romania, between 2022 and 2024, in order to evaluate the species as “One Health” sentinels at the wildlife–human–animal interface. During this period, 137 bacterial strains previously identified from 216 samples were phenotypically tested using both the Kirby–Bauer disk diffusion method and the Vitek 2 Compact system. Results: Among the Gram-negative isolates, particularly Escherichia coli and Salmonella enterica, notable antimicrobial resistance and multidrug-resistant (MDR) phenotypes were observed, including resistance to third-generation cephalosporins (ceftazidime) and reduced susceptibility to carbapenems. Resistance patterns observed in Proteus spp. largely reflected intrinsic resistance traits. Methicillin-resistant and MDR staphylococci (Staphylococcus aureus, S. pseudintermedius and S. sciuri) were detected in both peri-urban and peri-rural hunting grounds, with higher frequencies observed in peri-rural areas. Although MDR prevalence was slightly higher in peri-urban compared to peri-rural sites, no statistically significant association was identified between area of isolation and antimicrobial resistance or MDR status. Antimicrobial susceptibility results obtained by disk diffusion and the Vitek 2 Compact system showed a high level of concordance for antibiotics tested in common. Conclusions: Overall, these findings support the use of red foxes as effective One Health sentinels for monitoring environmental antimicrobial resistance occurrence across wildlife, domestic animals, and human-impacted habitats. Full article

Background/Objectives: Canine otitis externa (OE) is frequently complicated by Pseudomonas aeruginosa (P. aeruginosa) infections, which are often associated with treatment failure due to intrinsic and acquired antimicrobial resistance. This study aimed to assess the prevalence and antimicrobial susceptibility of P. aeruginosa isolates from dogs with OE in Timiș County, Romania, with a focus on aminoglycosides and fluoroquinolones, to provide region-specific, clinically relevant data and address potential public health implications. Methods: Exudate samples were collected from 435 dogs diagnosed with OE across multiple veterinary clinics between 2022 and 2025. P. aeruginosa isolates were identified using standard culture methods, and antimicrobial susceptibility was determined using the VITEK^® 2 Compact system according to CLSI VET01, Fifth Edition (2018) guidelines. Tested antibiotics included amikacin, gentamicin, enrofloxacin, marbofloxacin, and pradofloxacin. Resistance profiles were analyzed at both the individual antibiotic and class levels. Results:P. aeruginosa was isolated in 14.0% (61/435) of dogs. All isolates were susceptible to amikacin and gentamicin, whereas resistance to enrofloxacin and marbofloxacin was 27.9%, and pradofloxacin resistance reached 63.9%. A total of 24.6% of isolates were susceptible to all tested antibiotics. The most frequent multidrug-resistant combination among fluoroquinolones was ENR (R) + MAR (R) + PRA (R), observed in 23.0% of isolates. Conclusions: This study provides recent, region-specific data on P. aeruginosa prevalence and antimicrobial susceptibility in canine OE, offering clinically relevant insights into aminoglycoside and fluoroquinolone resistance. The findings highlight the potential public health significance of resistant P. aeruginosa strains at the human–animal interface and underscore the importance of antimicrobial stewardship in veterinary practice. Full article

Background/Objectives: Pseudomonas aeruginosa is an opportunistic pathogen frequently implicated in healthcare-associated infections, particularly ventilator-associated pneumonia and other device-related infections. The global emergence of carbapenem-resistant P. aeruginosa (CRPA) represents a major clinical challenge due to its limited therapeutic options and high mortality rates. Methods: Relevant clinical data were obtained from medical records. Isolates were identified via 16S PCR, and antimicrobial susceptibility testing was performed using the Vitek2 Compact system following CLSI guidelines. Carbapenemase genes (blaGES, blaKPC, blaIMP, blaNDM, blaVIM) were detected via PCR. Clonal relationships were determined via RAPD-PCR, and some sequence types were assigned according to the global P. aeruginosa MLST database. Results: In this study, 40 non-duplicate CRPA isolates were collected from 35 patients in a tertiary-care hospital in Mexico. Most isolates originated from adult patients, predominantly from tracheal aspirates (32.5%) and urine cultures (25.0%). Mechanical ventilation was the most common invasive device associated with infection, and the overall mortality rate reached 14.3%. Antimicrobial susceptibility testing showed that 95% of isolates exhibited a multidrug-resistant phenotype, with high resistance rates to ciprofloxacin (70.0%) and β-lactams. Carbapenemase genes were detected in 55% of isolates, mainly bla_IMP, bla_GES, and bla_VIM, either alone or in combination. Notably, this is the first report of ST309 (bla_IMP), ST411 (bla_GES + bla_IMP), and ST167 (bla_GES + bla_VIM) carrying carbapenemase genes in Mexico. Conclusions: These findings highlight the persistence and genetic diversity of CRPA circulating in hospital settings and emphasize the urgent need for strengthened genomic surveillance and infection control programs to prevent the spread of these high-risk multidrug-resistant clones. Full article

Fusidic acid resistance in Staphylococcus spp. has historically been confined to Staphylococcus ureilyticus, with limited data on its environmental distribution. This study presents the first detection of the fusidic acid resistance gene fusF in Staphylococcus kloosii recovered from virgin soil at Kampung Batu 16, Dusun Tua, Hulu Langat, Malaysia. A total of ten Staphylococcus isolates were identified using the VITEK^®2 system with high confidence (97–99%), comprising seven S. kloosii and three S. ureilyticus. Sequencing of representative isolates further corroborated the species identification. All isolates displayed phenotypic resistance to fusidic acid, while all S. ureilyticus (3/3) exhibited multi-drug resistant (MDR) traits and S. kloosii (7/7) exhibited non-MDR traits. PCR and sequencing confirmed the presence of fusF gene in S. ureilyticus (3/3) and S. kloosii (3/7). In addition, fusB and fusC genes were not detected in both species. The phylogenetic analysis (Maximum Likelihood, Tamura–Nei model) revealed high sequence conservation and clustering between fusF-positive S. kloosii and S. ureilyticus soil isolates, suggesting recent horizontal gene transfer between these two related species. The first detection of fusF gene in S. kloosii from virgin soil signifies the expansion of the ecological and host range beyond S. ureilyticus, establishes virgin soil as a potential antimicrobial resistance (AMR) reservoir, and underscores the One Health risks of resistance dissemination from environmental staphylococci. This baseline study highlights the importance of early AMR surveillance in tropical environments prior to agricultural development. Full article