Antimicrobial Resistance Genes Research Articles

Pulmonary infections in the elderly are difficult to diagnose due to immunosenescence, atypical symptoms, and frequent polymicrobial or opportunistic infections. Traditional methods often miss pathogens. Targeted next-generation sequencing (tNGS) enables rapid, sensitive, and comprehensive detection of pathogens and resistance genes, offering clear advantages. This retrospective study analyzed bronchoalveolar lavage fluid samples from 306 elderly inpatients with clinically suspected pulmonary infections using tNGS. Detected microbial species, antimicrobial resistance genes, and relevant clinical data were extracted from electronic medical records. Kaplan-Meier survival analysis and an eXtreme gradient boosting (XGBoost) machine learning model were employed to identify predictors of prolonged hospitalization. Viruses were the most frequently identified pathogens (29.6%), followed by bacteria (24.4%) and fungi (9.0%). Fungal co-infections were significantly associated with prolonged hospital stays and disrupted blood gas homeostasis, characterized by elevated partial pressure of carbon dioxide (PaCO₂) and bicarbonate (HCO₃⁻) levels. Antimicrobial resistance genes were detected in 7.5% of patients, with 23S rRNA mutations and mecA being the most prevalent. Inflammatory markers (C-reactive protein, procalcitonin) and gas exchange indices (PaCO₂, partial pressure of oxygen [PaO₂]) showed significantly correlations with overall pathogen burden. The eXtreme gradient boosting model identified PaCO₂, procalcitonin, HCO₃⁻, and fungal infection status as key predictors of prolonged hospitalization. tNGS facilitates comprehensive detection of pathogens and antimicrobial resistance genes in elderly patients with pulmonary infections. Our findings underscore the often overlooked clinical impact of fungal co-infection and respiratory dysfunction on patient outcomes. These results highlight the value of incorporating tNGS into routine diagnostic workflows for geriatric infection management, enhancing both diagnostic accuracy and prognostic assessment.

Escherichia coli are the reservoirs of antimicrobial resistance (AMR) genes. Shiga toxin-producing E. coli of animal origin can cause severe life-threatening infections in man. The present study investigated the susceptibility of poultry-origin E. coli isolates to Clinical and Laboratory Standard Institute (CLSI)-recommended antibiotics and their Shiga toxin profiles. A total of 141 samples comprising cloacal swabs and water and litter samples were investigated. Antimicrobial susceptibility profiles of the recovered isolates were determined for 15 antibiotics as recommended by CLSI. Shiga toxin-encoding genes (Shiga toxin 1 [stx1] and Shiga toxin 2 [stx2]) and plasmid-mediated AMR determinants were ascertained by the polymerase chain reaction. E. coli isolation rates were 90.1% with a higher incidence in organized (93.8%) compared to backyard rearing systems (87%). AMR (21.3%) was limited to only three antibiotic classes, i.e., penicillins (9.4%), fluoroquinolones (11.0%), and phenicols (0.8%). All the isolates were susceptible to aminoglycosides, carbapenems, monobactams, and polymyxins with the absence of multidrug resistance. The incidence of resistant E. coli was significantly higher in organized rearing systems (30.0%) compared to backyard units (13.4%). All penicillin-resistant isolates were positive for blaSHV and two isolates co-expressed blaOXA-1 with blaSHV. 21.4% of fluoroquinolone-resistant isolates were positive for plasmid-mediated aac (6')-Ib resistance. 96.3% of resistant isolates carried isolates stx2 and none had stx1. Low AMR incidence was recorded in E. coli isolates of poultry origin for CLSI-recommended old and newer classes of antibiotics. Higher levels of stx2 detections in antimicrobial-resistant E. coli isolates pose a significant public health threat.

Long-read metagenomic sequencing allows for the rapid, culture-independent, and accurate identification of causative pathogens and antimicrobial resistance (AMR) profiles, supporting precise antibiotic use and reducing the spread of resistance. However, its application to mastitis milk is challenging due to the complex milk matrix, low bacterial count, and high somatic cell content. This study primarily aimed to further optimize our previously developed culture-free nanopore sequencing protocol for milk samples from mastitis cases. Additional optimizations included combining centrifugation, gradient centrifugation, and fat fraction treatment with Tween 20 and citric acid. Subsequently, four DNA extraction kits (Blood and Tissue, Molysis Complete5, HostZero, and SPINeasy Host depletion) were evaluated for their ability to remove host DNA and enrich bacterial DNA for long-read sequencing with Oxford Nanopore technologies. qPCR was used to quantify bacterial and bovine DNA, allowing comparison of host depletion efficiency among the kits. Our results show that simple centrifugation effectively concentrates bacterial cells, removing the need for chemical treatments. The HostZero kit consistently produced higher DNA yields, improved DNA integrity, and more effective host DNA depletion. Using nanopore sequencing, both Gram-positive and Gram-negative mastitis pathogens, along with their AMR genes, were successfully detected. Overall, this study underscores the importance of an effective DNA extraction method for the direct sequencing of mastitis milk samples. Additionally, our findings support the potential of direct metagenomic sequencing as a rapid, culture-free approach for identifying mastitis pathogens and their resistance profiles.

There has been an increased interest in phage therapy for the treatment of antimicrobial resistant bacterial infections due to the decreasing effectiveness of antibiotics. While phage therapy has been successful, it often requires time to isolate and characterize phages suitable for phage therapy. Here, we isolated and characterized three phages against carbapenem-resistant Enterobacter cloacae complex (ECC) strains to potentially be applied in phage therapy. ECC strains commonly cause sepsis and urinary tract infections in hospitals and are designated as a pathogen of critical importance for its global prevalence and high instance of antimicrobial resistance. Initially, a total of 96 phages were isolated using a collection of diverse clinical ECC strains. Three phages were selected for genome sequencing and additional characterization based on their broad host range and lytic activity. Based on average nucleotide identity (ANI), vB_ECC_YI837 and vB_ECC_MY742 were considered strains of previously isolated phages vB_CsaM_leN and Entb_45, respectively. One phage had an ANI of 92.65% and was therefore considered novel and named vB_ECC_CW742. Furthermore, none of the phages carried toxin, antimicrobial resistance, or lysogeny-related genes. Collectively, the phages covered 82% (108/131) of our carbapenem-resistant ECC strain library. These findings suggest that the three phages are promising candidates for phage therapy against carbapenem-resistant ECC infections.

Background/Objectives: Long-read metagenomic sequencing can detect bacteria and antimicrobial resistance genes (ARGs) from bovine respiratory samples, providing an alternative to culture and antimicrobial susceptibility testing (C/S). This study applied Bayesian latent class models (BLCMs) to estimate the sensitivity (Se) and specificity (Sp) of long-read metagenomic sequencing compared to C/S for detecting Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni, as well as associated macrolide and tetracycline resistance potential. Methods: Deep nasopharyngeal swabs were collected from fall-placed feedlot calves at arrival, 13, and 36 days on feed across two years and two metaphylaxis protocols. Samples underwent C/S and long-read metagenomic sequencing. BLCMs were used to estimate Se and Sp for the detection of bacteria and potential for antimicrobial resistance (AMR). Results: Se and Sp for detecting respiratory bacteria by metagenomics were not significantly different than culture, with four exceptions. For the 2020 samples, Se for M. haemolytica was lower than culture, and Sp for H. somni was lower, while in both 2020 and 2021 samples, Se for P. multocida was higher for metagenomics than culture. The estimated Se and Sp of metagenomics for the detection of msrE-mphE, EstT, and tet(H) within bacterial reads were either not significantly different or were lower than AST, with Sp > 95% with one exception. Conclusions: This study provided BLCM-based estimates of clinical Se and Sp of metagenomics and C/S without assuming a gold standard in a large pen research setting. These findings demonstrate the potential of long-read metagenomics to support bovine respiratory disease diagnostics, AMR surveillance, and antimicrobial stewardship in feedlot cattle.

An increase of group B Streptococcus (GBS) colonization in pregnant women with a parallel rise in neonatal and infant infections, were observed in Korea. We characterized antimicrobial resistance (AMR) and molecular features of GBS isolates from reproductive-aged women between 1994-2000 and 2017-2022. We collected 246 GBS isolates, 37 during 1994-2000 and 209 during 2017-2022, from cervical and/or anorectal swabs at three institutions. Antimicrobial susceptibility was tested using the MicroScan MicroSTREP Plus Panel (Beckman Coulter, Brea, CA, USA). Sequence types (STs), clonal complexes (CCs), cps genotypes by serotypes, and AMR genes were identified using whole-genome sequencing on the NovaSeq 6000 system (Illumina, San Diego, CA, USA). During 1994-2000, CC19 was predominant (35.1%, 13/37), whereas during 2017-2022, CC1 became the most common (35.4%, 74/209). cps genotype VIII, previously limited to one ST1 isolate, appeared in 32 ST2 isolates (P =0.037). All isolates remained susceptible to β-lactams and vancomycin. Tetracycline resistance decreased from 97.3% to 60.8% (P <0.001), with tetM prevalence decreasing from 91.7% to 72.4% (P <0.001) and tetO prevalence increasing from 2.8% to 29.9% (P =0.017). Levofloxacin resistance increased from 0% to 23.4% (P =0.001), with 98.0% of resistant isolates carrying both gyrA and parC. The number of resistance profiles increased from six to 16, including 11 newly identified patterns, covering 81.8% of levofloxacin resistant isolates. The acquisition of diverse resistance genes has expanded AMR profiles in colonized GBS, emphasizing the need for sustained nationwide surveillance.

BackgroundChronic infections in cystic fibrosis (CF) patients are often driven by adaptive and drug-resistant bacterial lineages. Understanding sequence types (STs) and phenotypic adaptations such as small colony variants (SCVs) can guide more effective clinical management.MethodsSeventeen bacterial isolates from respiratory specimens of 12 pediatric CF patients (Dnipro, Ukraine, Sept 2022–Sept 2024) were characterized via culture-based diagnostics and whole genome sequencing (WGS). Multi-Locus Sequence Typing (MLST) was applied to determine sequence types, while phenotypic traits, including SCV morphology, were recorded and antimicrobial resistance (AMR) genes profiled.ResultsThe collection included Staphylococcus aureus (n = 8), Pseudomonas aeruginosa (n = 5), Serratia marcescens (n = 2), Enterobacter hormaechei (n = 1), and Klebsiella pneumoniae (n = 1). SCV phenotypes were observed in 2 S. aureus ST398 isolates. P. aeruginosa strains belonged to diverse STs (ST644, ST2967, ST1228, ST242). The hypervirulent K. pneumoniae ST23-KL1 isolate harbored siderophores (iucA, ybt) and peg-344. All S. aureus ST398 isolates exhibited reduced growth rate and dense colony morphology.ConclusionThis study highlights the genetic and phenotypic complexity of respiratory pathogens in Ukrainian CF children, emphasizing the emergence of ST398 SCVs and the introduction of hypervirulent K. pneumoniae lineages. The findings call for regular genomic surveillance in Ukrainian CF cohorts.

We present draft genomes of Citrobacter freundii strains isolated from a model wastewater treatment plant seeded with sludge from Stillwater, Oklahoma. Analysis suggests that the Stillwater strains are related to human isolates from China and the UK. All strains also possess antimicrobial resistance genes, highlighting the potential public health implications.

Objectives: Studying contemporary antimicrobial resistance ( AMR ) gene distribution in Staphylococcus aureus through comprehensive genomic surveillance analysis by characterizing the prevalence, distribution, geographical context, and co-occurrence of AMR genes in a large collection of recent S. aureus genomes. Materials and Methods: A retrospective genomic surveillance study was conducted on 1,152 complete S. aureus genomes from the National Center for Biotechnology Information RefSeq database with release dates between January 2020 and April 2025. AMR genes were identified using AMRFinderPlus. Statistical analysis: Custom Python scripts using specialized libraries for data manipulation and analysis, supplemented by R statistical software for advanced genomic epidemiology analyses, were performed to determine AMR gene prevalence, distribution, and co-occurrence relationships. Results: An overwhelming 94.27% (1,086/1,152, 95% confidence interval [92.78, 95.47]) of genomes harbored at least one AMR gene. The analysis identified 135 unique AMR genes across 25 drug classes. The tetracycline resistance gene, tet(38) , was present in 99.91% of AMR-positive genomes. The methicillin resistance gene, mecA , was detected in 50.18% of AMR-positive isolates. Acquired resistance genes were 3.3 time s more frequent than point mutations. Analysis of geographical metadata revealed high AMR rates globally, though this study provides a genotypic prediction of resistance, without phenotypic correlation, and may be influenced by sampling bias inherent in public databases, which primarily contain clinically submitted genomes. Conclusions: This study reveals a high prevalence and complex architecture of AMR genes in contemporary S. aureus populations. These findings highlight the need for enhanced global genomic surveillance to mitigate the threat of multidrug-resistant S. aureus .

Background/Objectives: Hospital wastewater is a complex effluent containing a wide range of biological and chemical contaminants, including pharmaceuticals, pathogens, and antimicrobial resistance determinants. These discharges pose a growing threat to aquatic ecosystems and public health, particularly in regions where wastewater treatment is insufficient. This study aimed to characterize the chemical and microbiological composition of untreated effluent from a tertiary care hospital in southern Chile, focusing on contaminants of emerging concern. Methods: Wastewater samples were collected at the hospital outlet before any treatment. The presence of two commonly used pharmaceutical compounds, paracetamol and amoxicillin, was quantified using high-performance liquid chromatography (HPLC). Bacterial isolation was performed using selective media, and antibiotic susceptibility testing was conducted via the disk diffusion method following CLSI guidelines. In addition, metagenomic DNA was extracted and sequenced to assess microbial community composition and functional gene content, focusing on the identification of resistance genes and potential pathogens. Results: A total of 42 bacterial isolates were recovered, including genera with known pathogenic potential such as Aeromonas, Klebsiella, and Enterococcus. Antibiotic susceptibility tests revealed a high prevalence of multidrug-resistant strains. Metagenomic analysis identified the dominance of Bacillota and Bacteroidota, together with 56 antimicrobial-resistance gene (ARG) families and 38 virulence-factor families. Functional gene analysis indicated the presence of efflux-pump systems, β-lactamases, and mobile genetic elements, suggesting that untreated hospital effluents serve as potential sources of resistance and virulence determinants entering the environment. Paracetamol was detected in all samples, with an average concentration of 277.4 ± 10.7 µg/L; amoxicillin was not detected, likely due to its instability and rapid degradation in the wastewater matrix. Conclusions: These findings highlight the complex microbiological and chemical burden of untreated hospital wastewater and reinforce the need for continuous monitoring and improved treatment strategies to mitigate environmental dissemination of antibiotic resistance.

Guangdong Province is an important area of poultry breeding in China. Zhanjiang city is located in the western part of Guangdong Province, where there are many broiler farms. To investigate antimicrobial resistance (AMR) and the presence of resistance genes in Escherichia coli from broiler farms, a total of 220 samples were collected from soil and feces at eight broiler farms. Subsequently, 220 strains of E. coli were isolated for drug resistance analysis and detection of AMR genes. The results revealed that the isolated E. coli strains exhibited high prevalence of multidrug resistance to 12 antimicrobial drugs including amoxicillin, tetracycline, cotrimoxazole and sulfisoxazole. Among the isolated strains, 95% of the isolates were resistant to more than three antimicrobial agents; notably, thirty-nine strains showed multidrug resistance to ten tested drugs, while four strain exhibits multidrug resistance to as many as fifteen antibacterial drugs. Additionally, seven AMR genes such as blaTEM and sul2 were detected in over half (≥50%) of the isolated E. coli samples; thirteen AMR genes had relatively low detection prevalence (≤30%). Correlation analysis indicated a strong association between certain AMR genes (blaTEM, pexA, aadA1, blaAIM, ant(3")-I, sul2, sul3, tet(D)) and AMR (≥50%). In conclusion, E.coli strains obtained from soil and fecal samples in broiler farms exhibited multidrug resistant phenotypes along with carrying various AMR genes. This provides a reference for the scientific control of E. coli multidrug resistance in this area.

Wastewaters are considered as hotspot for multidrug resistant bacteria and genes, especially for extended spectrum beta lactamase producing Escherichia coli (ESBL-EC), which pose significant public health concern. Conventional wastewater treatment plants (WWTPs) are not designed to remove such resistant bacteria before discharge. This study, therefore, aimed to evaluate and compare the performance of two municipal WWTPs in eliminating ESBL-EC in Hatay province, Türkiye. The isolates were further characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and resistance gene analysis. A total of 24 wastewater samples [influent (n = 12) and effluent (n = 12)] were collected from both conventional and advanced biological WWTP. A total of 66 ESBL-EC were isolated and confirmed as ESBL producer. The abundance of ESBL-EC was significantly more prevalent in the influent water samples (mean 3.86 ± 0.11 log cfu/ml), when compared to the effluent wastewater samples (mean 2.20 ± 0.27 log cfu/ml). According to the PFGE results (cut-off > 85%), 16 isolates were clonally related, whereas 49 isolates were singletons and one isolate could not be evaluated. Besides beta-lactam antibiotics, the isolates were highly resistant to tetracycline (63.63%) and fluoroquinolones (43.93%). The blaCTX-M gene was the most frequently detected gene in the isolates (86.36%), among which the blaCTX-M-15 type (86.36%) was the most frequently detected. At least two disinfectant resistance genes were also detected in each of the isolates. Even though both WWTP were found to be effective in reducing ESBL-EC counts, not well enough to complete elimination, showing the potential public health risk.

Diagnosing urinary tract infections (UTIs) in older women is challenging due to the high prevalence of asymptomatic bacteriuria (ASB). This study aimed to investigate whether pathogen-related factors differ between older women with UTI and those with ASB, and to gain further insight into the persistence of Escherichia coli in ASB. In this exploratory study, E. coli isolates from 46 participants were analysed. The study included community-dwelling and long-term care facility women aged ≥ 65years diagnosed with either UTI (defined as ≥ 2 new-onset lower urinary tract symptoms, pyuria, and bacteriuria) or ASB (E. coli ≥ 104CFU/mL in two consecutive urine samples). Whole-genome sequencing (WGS) was performed to identify previously described putative uropathogenicity factors (PUFs), antimicrobial resistance genes, multilocus sequence typing (MLST) sequence types (STs), and Clermont phylogroups. We analysed 31 isolates from UTI patients and 32 sequential isolates from 15 ASB patients. All ASB patients carried genetically similar E. coli strains in two consecutive samples taken 2-4weeks apart. The isolates represented a diverse range of STs, with ST69, ST73, and ST141 being the most prevalent. Although no significant difference in the overall number of PUFs between UTI and ASB groups was observed, distinct patterns emerged. UTI-associated isolates more frequently harbored cnf1 and sfaH, whereas iha, iucC and sat were more common in ASB isolates. Our findings suggest potential individual differences in the presence of PUFs between UTI and ASB isolates. Further functional studies are warranted to explore the role of these factors in bladder colonization and UTI pathogenesis. Additionally, their interactions with host-specific factors should be examined to better understand bacterial persistence and disease development in older women.

Venomous snakes constitute ecologically significant and medically relevant organisms due to the risks associated with their bites, which frequently result in secondary infections. The oral microbiota of these reptiles plays a crucial role in the pathogenesis of such infections; however, its diversity and clinical implications remain insufficiently characterized. This is the first comprehensive review to systematically trace the methodological evolution in snake oral microbiota research, documenting the paradigm shift from traditional culture-dependent techniques to advanced culture-independent approaches, including next-generation sequencing and metagenomics. Our analysis uniquely demonstrates the transformative impact of these technological advances on bacterial diversity identification and antimicrobial resistance gene detection in venomous species. Environmental factors, captivity conditions, and venom composition significantly influence microbial community structure and resistance profiles. These intricate interactions are essential for improving clinical management of snakebite infections, informing empirical antibiotic therapy protocols, and guiding antivenom production strategies. Additionally, the potential of snake oral microbiota as a source of novel bioactive compounds represents an emerging area of bioprospecting research. This review uniquely bridges microbiology, venomics, and clinical medicine, demonstrating the necessity for integrative, multidisciplinary approaches to fully elucidate the ecological and biomedical significance of oral microbial communities in venomous snakes.

Background/Objectives: This study characterized the phenotypic and genotypic profiles of antimicrobial resistance in 104 Escherichia coli isolates obtained from 22 samples of artisanal Minas Frescal cheese from the Federal District, Brazil. Methods: The antimicrobial susceptibility of E. coli isolates was assessed using the disk diffusion method and antimicrobial resistance genes were detected using polymerase chain reaction methods with specific primers. Results: The highest rates of phenotypic antimicrobial resistance were observed for sulfonamides (85.58%, 89/104) and tetracyclines (38.46%, 40/104). In the genotypic profiles, most E. coli isolates carried the sulfonamide resistance genes sul1/sul2 (62.50%, 65/104), tetracycline resistance genes tetA/tetB (65.38%, 68/104), and β-lactam resistance genes blaCTX-M/blaTEM/blaSHV (55.77%, 58/104). Most E. coli strains that presented sulfonamide resistance genes carried the sul1 gene (49.04%, 51/104) and were phenotypically sulfonamide-resistant strains (59.61%, 62/104). Regarding the E. coli strains that carried tetracycline resistance genes, the majority harbored both tetA and tetB genes (34.61%, 36/104), with 35.56% (37/104) being phenotypically resistant and 29.80% (31/104) being phenotypically susceptible. For E. coli strains that presented β-lactam resistance genes, the most frequently detected gene was blaCTX-M (21.15%, 22/104) and, notably, most E. coli strains (43.26%, 45/104) were phenotypically susceptible. The cat1 and clmA genes (associated with phenicol resistance) were detected in 22.12% of the E. coli isolates (23/104), with only two strains (1.92%) being phenotypically resistant to chloramphenicol. Conclusion: The high prevalence of E. coli carrying antimicrobial resistance genes in artisanal cheese raises public health concerns regarding the dissemination of potentially pathogenic antimicrobial-resistant microorganisms through the food chain.

Shellfish are an essential component of the human diet, yet their safety is increasingly compromised by contamination with heavy metals, petroleum hydrocarbons, and pathogenic microorganisms, such as Vibrio, which pose significant health risks. This study examined shellfish samples from seafood markets, assessing the levels of heavy metals (e.g., cadmium, copper) and petroleum hydrocarbons, while isolating and identifying Vibrio species carried by the shellfish. The antimicrobial resistance profiles, resistance genes, and biofilm-forming capacities of these strains were further characterized. Results revealed significant seasonal fluctuations in heavy metal concentrations, with some samples exceeding regulatory limits, indicating potential health risks for long-term consumers. Likewise, Vibrio abundance and resistance varied seasonally, with a notable prevalence of multidrug-resistant strains, likely influenced by antibiotic misuse and environmental pressures in coastal regions. Correlation analyses suggested potential links between heavy metal contamination and Vibrio resistance, as well as biofilm formation, supporting the hypothesis that metal-induced stress may facilitate resistance gene transfer and enhance biofilm-mediated resistance. This study reveals the seasonal dynamics of antimicrobial resistance (AMR) in shellfish-derived Vibrio species and elucidates the dose–response effects of heavy metals and petroleum hydrocarbons, as well as their synergistic selection mechanisms. These findings provide a scientific foundation for assessing shellfish safety, deciphering AMR transmission, and developing ecosystem-based strategies for aquaculture monitoring.

Exploring the indoor airborne microbiome and resistome in layer barns across Alberta, Canada.

Genetic comparison of virulence and resistance factors in phylogenetically diverse Escherichia coli from dairy cattle.

Urban trash bins as reservoirs of multidrug-resistant Enterococcus with shared resistance genes across clinical and environmental niches.

Comprehensive whole-genome analysis of Streptococcus infantarius strains from Moroccan farmhouse dairy products: Genomic insights into dairy adaptation, safety, and biotechnological potential.