Antimicrobial Resistance Genes Research Articles

Evaluating Sequence Alignment Tools for Antimicrobial Resistance Gene Detection in Assembly Graphs

Antimicrobial resistance (AMR) is an escalating global health threat, often driven by the horizontal gene transfer (HGT) of resistance genes. Detecting AMR genes and understanding their genomic context within bacterial populations is crucial for mitigating the spread of resistance. In this study, we evaluate the performance of three sequence alignment tools—Bandage, SPAligner, and GraphAligner—in identifying AMR gene sequences from assembly and de Bruijn graphs, which are commonly used in microbial genome assembly. Efficiently identifying these genes allows for the detection of neighboring genetic elements and possible HGT events, contributing to a deeper understanding of AMR dissemination. We compare the performance of the tools both qualitatively and quantitatively, analyzing the precision, computational efficiency, and accuracy in detecting AMR-related sequences. Our analysis reveals that Bandage offers the most precise and efficient identification of AMR gene sequences, followed by GraphAligner and SPAligner. The comparison includes evaluating the similarity of paths returned by each tool and measuring output accuracy using a modified edit distance metric. These results highlight Bandage’s potential for contributing to the accurate identification and study of AMR genes in bacterial populations, offering important insights into resistance mechanisms and potential targets for mitigating AMR spread.

Strategy to develop and validate digital droplet PCR methods for global antimicrobial resistance wastewater surveillance.

According to World Health Organization (WHO), antimicrobial resistance (AMR) is currently one of the world's top 10 health threats, causing infections to become difficult or impossible to treat, increasing the risk of disease spread, severe illness, disability, and death. Accurate surveillance is a key component in the fight against AMR. Wastewater is progressively becoming a new player in AMR surveillance, with the promise of a cost-effective real-time tracking of global AMR profiles in specific regions. One of the most useful analytical methods for wastewater surveillance is currently based on real-time PCR (qPCR) and digital droplet PCR (ddPCR) technologies. As stated in the EU Wastewater Treatment Directive proposal, methodological standardization, including a workflow for method development and validation, will play a crucial role in global monitoring of AMR in wastewater. However, according to our knowledge, there are currently no qPCR and ddPCR methods for AMR surveillance available that have been validated according to international standard performance criteria. Therefore, this study proposes a workflow for the development and validation of PCR-based methods for a harmonized and global AMR surveillance, including the construction of specific sequence databases and microbial collections for an efficient method development and method specificity evaluation. Following this strategy, we have developed and validated four duplex ddPCR methods responding to international standard performance criteria, focusing on seven AMR genes (ARG's), including extended spectrum beta-lactam (blaCTX-M), carbapenem (blaKPC-2/3), tetracycline (tet(M)), erythromycin (erm(B)), vancomycin (vanA), sulfonamide (sul2), and aminoglycoside (aac(3)-IV), as well as one indicator of antibiotic (multi-) resistance and horizontal gene transfer, named the class I integron (intl1). The performance of these ddPCR methods was successfully assessed for their specificity, as no false-positive and false-negative results were observed. These ddPCR methods were also considered to be highly sensitive as showing a limit of detection below 25 copies of the targets. In addition, their applicability was confirmed using 14 wastewater samples collected from two Belgian water resource recovery facilities. The proposed study represents therefore a step forward to reinforce method harmonization in the context of the global AMR surveillance in wastewater. PRACTITIONER POINTS: In the context of wastewater surveillance, no PCR-based methods for global AMR monitoring are currently validated according to international standards. Consequently, we propose a workflow to develop and validate PCR-based methods for a harmonized and global AMR surveillance. This workflow resulted here in four duplex ddPCR methods targeting seven ARGs and one general indicator for mobilizable resistance genes. The applicability of these validated ddPCR methods was confirmed on 14 wastewater samples from two Belgian water resource recovery facilities.

Virulence characterization of Staphylococcus spp. isolated from mozzarella cheeses and cheese-slicers

Different species of the Staphylococcus genus can be found as contaminants in foods and may carry virulence factors that could potentially make them The enterotoxigenic potential ofpathogenic or transfer genes to other bacteria. Therefore, the aim of the present study was to isolate and identify species of Staphylococcus genus isolated from sliced mozzarella cheeses and cold-cut slicers and characterize biofilm formation in vitro, presence of enterotoxins and antibiotic resistance genes, and β-lactamase production. Sampling was carried out in 44 retail markets and isolates identified on species level. Resistance genes blaZ and mecA, as well as enterotoxin genes sea, seb, sec, and sed, were identified using PCR. Phenotypic evaluation of biofilm formation and detection of β-lactamase enzyme were also analyzed. Nine species of coagulase-negative Staphylococcus (CNS) were identified from 85 bacterial isolates. Of this total, 67.0% were positive for the presence of one or both antimicrobial resistance genes blaZ and mecA, while the β-lactamase enzyme was detected in 52.9% isolates. Regarding biofilm production, 48.2% of isolates were positive and 51.8% were negative. However, none of the isolates were identified with the enterotoxin genes. The results demonstrate the potential risk of cross-contamination, as biofilm-producing CNS could persist in the cheese retail environment, highlighting the danger of resistance genes transferring to other commensal or pathogenic bacteria.

Whole-Genome Sequencing Analysis of Antimicrobial Resistance, Virulence Factors, and Genetic Diversity of Salmonella from Wenzhou, China

Salmonella species are important foodborne pathogens worldwide. Salmonella pathogenicity is associated with multiple virulence factors and enhanced antimicrobial resistance. To determine the molecular characteristics and genetic correlations of Salmonella, 24 strains of Salmonella isolated from different sources (raw poultry, human stool, and food) in the Wenzhou area were investigated to determine the distribution of antimicrobial resistance and virulence determinants using whole-genome sequencing (WGS). Aminoglycoside resistance genes were detected in all samples. Over half of the samples found antimicrobial resistance genes (ARGs) and point mutations for several clinically frequently used antibiotic, beta-lactams, tetracyclines, and quinolones. Of these strains, 62.5% were predicted to be multidrug-resistant (MDR). The quinolone-modifying enzyme gene aac(6’)-Ib-cr, detected in five samples (S1–S4 and S10), was located on integrons. The analysis of Salmonella pathogenicity island (SPI) profiles suggests that serotypes with close genetic relationships share the same distribution of virulence factors, revealing a link between genotype and SPI profiles. cgMLST analysis indicated that five isolates S14–S18 were closely related to strains originating from the United Kingdom, suggesting that they may share a common origin. Data from this study may enrich the molecular traceability database for Salmonella and provide a basis for effective public health policies.

PIPdb: a comprehensive plasmid sequence resource for tracking the horizontal transfer of pathogenic factors and antimicrobial resistance genes.

Plasmids, as independent genetic elements, carrying resistance or virulence genes and transfer them among different pathogens, posing a significant threat to human health. Under the 'One Health' approach, it is crucial to control the spread of plasmids carrying such genes. To achieve this, a comprehensive characterization of plasmids in pathogens is essential. Here we present the Plasmids in Pathogens Database (PIPdb), a pioneering resource that includes 792964 plasmid segment clusters (PSCs) derived from 1 009571 assembled genomes across 450 pathogenic species from 110 genera. To our knowledge, PIPdb is the first database specifically dedicated to plasmids in pathogenic bacteria, offering detailed multi-dimensional metadata such as collection date, geographical origin, ecosystem, host taxonomy, and habitat. PIPdb also provides extensive functional annotations, including plasmid type, insertion sequences, integron, oriT, relaxase, T4CP, virulence factors genes, heavy metal resistance genesand antibiotic resistance genes. The database features a user-friendly interface that facilitates studies on plasmids across diverse host taxa, habitats, and ecosystems, with a focus on those carrying antimicrobial resistance genes (ARGs). We have integrated online tools for plasmid identification and annotation from assembled genomes. Additionally, PIPdb includes a risk-scoring system for identifying potentially high-risk plasmids. The PIPdb web interface is accessible at https://nmdc.cn/pipdb.

Assessment of Bacterial Contamination and Antimicrobial Resistance of Escherichia coli Isolates from Slovak Dairy Farms

The conditions in livestock housing are suitable for the survival of airborne microorganisms, mainly due to high temperatures, humidity, and the presence of organic material. The total count of airborne bacteria concentrations in cattle farms ranged from 3.01 log10 CFU/mL to 6.90 log10 CFU/mL; for coliform bacteria, they were from 2.18 log10 CFU/mL to 3.34 log10 CFU/mL; and for molds, they ranged from 3.00 log10 CFU/mL to 4.57 log10 CFU/mL. Bacteria resistant to antimicrobial substances and resistance genes can be spread on animal farms. Antimicrobial resistance in ubiquitous Escherichia coli isolated from cattle feces was investigated. Minimum inhibitory concentration (MIC) testing was utilized to identify phenotypic resistance profiles, and the PCR method was employed to detect the presence of resistant genes. A higher percentage of resistance was found to amikacin (65%), tetracycline (61%), streptomycin (56%), ampicillin (55%), and nalidixic acid (45%). Multidrug resistance was determined in up to 64.3% of the isolates studied. The most widespread resistance genes were blaTEM (85.7%), sul2 (66.7%), tetB (52.38%), and sul1 (47.6%). We found that 4.8% of the E. coli isolates had the blaCMY gene. We found that, despite phenotypic resistance, E. coli isolates do not necessarily carry genes conferring resistance to that particular antimicrobial agent.

Comparison of direct cDNA and PCR-cDNA Nanopore sequencing of RNA from Escherichia coli isolates.

Whole-transcriptome (long-read) RNA sequencing (Oxford Nanopore Technologies, ONT) holds promise for reference-agnostic analysis of differential gene expression in pathogenic bacteria, including for antimicrobial resistance genes (ARGs). However, direct cDNA ONT sequencing requires large concentrations of polyadenylated mRNA, and amplification protocols may introduce technical bias. Here we evaluated the impact of direct cDNA- and cDNA PCR-based ONT sequencing on transcriptomic analysis of clinical Escherichia coli. Four E. coli bloodstream infection-associated isolates (n=2 biological replicates per isolate) were sequenced using the ONT Direct cDNA Sequencing SQK-DCS109 and PCR-cDNA Barcoding SQK-PCB111.24 kits. Biological and technical replicates were distributed over eight flow cells using 16 barcodes to minimize batch/barcoding bias. Reads were mapped to a transcript reference and transcript abundance was quantified after in silico depletion of low-abundance and rRNA genes. We found there were strong correlations between read counts using both kits and when restricting the analysis to include only ARGs. We highlighted that correlations were weaker for genes with a higher GC content. Read lengths were longer for the direct cDNA kit compared to the PCR-cDNA kit whereas total yield was higher for the PCR-cDNA kit. In this small but methodologically rigorous evaluation of biological and technical replicates of isolates sequenced with the direct cDNA and PCR-cDNA ONT sequencing kits, we demonstrated that PCR-based amplification substantially improves yield with largely unbiased assessment of core gene and ARG expression. However, users of PCR-based kits should be aware of a small risk of technical bias which appears greater for genes with an unusually high (>52%)/low (<44%) GC content.

How Gut Microbiome Perturbation Caused by Antibiotic Pre-Treatments Affected the Conjugative Transfer of Antimicrobial Resistance Genes

The global spread of antimicrobial resistance genes (ARGs) poses a significant threat to public health. While antibiotics effectively treat bacterial infections, they can also induce gut dysbiosis, the severity of which varies depending on the specific antibiotic treatment used. However, it remains unclear how gut dysbiosis affects the mobility and dynamics of ARGs. To address this, mice were pre-treated with streptomycin, ampicillin, or sulfamethazine, and then orally inoculated with Salmonella enterica serovar Typhimurium and S. Heidelberg carrying a multi-drug resistance IncA/C plasmid. The streptomycin pre-treatment caused severe microbiome perturbation, promoting the high-density colonization of S. Heidelberg and S. Typhimurium, and enabling an IncA/C transfer from S. Heidelberg to S. Typhimurium and a commensal Escherichia coli. The ampicillin pre-treatment induced moderate microbiome perturbation, supporting only S. Heidelberg colonization and the IncA/C transfer to commensal E. coli. The sulfamethazine pre-treatment led to mild microbiome perturbation, favoring neither Salmonella spp. colonization nor a conjugative plasmid transfer. The degree of gut dysbiosis also influenced the enrichment or depletion of the ARGs associated with mobile plasmids or core commensal bacteria, respectively. These findings underscore the significance of pre-existing gut dysbiosis induced by various antibiotic treatments on ARG dissemination and may inform prudent antibiotic use practices.

Multidrug-resistant Salmonella Typhi among symptomatic and asymptomatic children in informal settlements in Nairobi, Kenya

BackgroundThe emergence and persistence of multidrug-resistant (MDR) Salmonella Typhi (S. Typhi) infections is a significant global health problem. The carrier state of typhoid makes it prudent to conduct routine surveillance for both acute cases and carriers especially those caused by MDR S. Typhi. We report on the prevalence of MDR S. Typhi, resistance phenotypes and antimicrobial resistance genes detected in symptomatic and asymptomatic children living in informal settlements in Nairobi, Kenya.Methods215 archived presumed S. Typhi isolates from stool samples provided by children ≤ 16 years collected from 2013 to 2018 were revived in May, 2022 and confirmed using culture and antisera serotyping. The Kirby Bauer disc diffusion technique was used to test the S. Typhi against 14 antibiotics. The MDR S. Typhi (resistant to ampicillin, chloramphenicol and sulfamethoxazole trimethoprim) which in addition were also resistant to either a cephalosporin or a fluoroquinolone were analyzed for Beta lactams and quinolone resistance genes using polymerase chain reaction.ResultsA total of 215 isolates were confirmed to be positively S. Typhi; of these, 105 (49%) and 110 (51%) were from symptomatic and asymptomatic children respectively. On average, S. Typhi resistance from asymptomatic and symptomatic children against 1st line drugs was observed at; 77% &70%, ampicillin; 60% & 64%, sulfamethoxazole-trimethoprim, and 45% & 54%, chloramphenicol respectively. Multi drug resistance was observed in 90 (42%) of the isolates, of these, 44 (49%) were isolated from symptomatic and 46 (51%) from asymptomatic children. Fifteen resistance phenotypes (p) were observed with, ampicillin/chloramphenicol/sulfamethoxazole-trimethoprim/nalidixic acid (amp/chl/sxt/na) as the most common among the symptomatic 43/90 (48%) and asymptomatic 55/90 (61%) children. The blaTEM−D, AMR genes were detected in 37/44 (84%) S. Typhi isolates, out of this 18 (49%) were from symptomatic while 19 (51%) were from asymptomatic children respectively.ConclusionThe carriage of MDR S. Typhi among the asymptomatic children is concerning as they can act as potential transmitters of the typhoid disease to unsuspecting children. These study findings highlight the need for continued surveillance of antimicrobial resistance and mass immunization of children living in these urban informal areas.

Isolation and Characterization of a Novel Jumbo Phage HPP-Temi Infecting Pseudomonas aeruginosa Pa9 and Increasing Host Sensitivity to Ciprofloxacin

Pseudomonas aeruginosa is a bacteria responsible for many hospital-acquired infections. Phages are promising alternatives for treating P. aeruginosa infections, which are often intrinsically resistant. The combination of phage and antibiotics in clearing bacterial infection holds promise due to increasing reports of enhanced effectiveness when both are used together. The aim of the study is to isolate and characterize a novel P. aeruginosa phage and determine its effectiveness in in vitro combination with antibiotics in controlling P. aeruginosa. In this study, a novel jumbo myophage HPP-Temi infecting P. aeruginosa Pa9 (PP334386) was isolated from household sewage. Electron micrographs of the phage were obtained to determine the morphological features of HPP-Temi virions. Complete genome analysis and a combination of Pseudomonas phage HPP-Temi with antibiotics were examined. The phage HPP-Temi was able to productively infect P. aeruginosa ATCC 9027 but was unable to infect a closely related genus. The phage was stable at 4–37 °C, 0.5% NaCl, and pH 8 for at least one hour. The HPP-Temi genome is a 302,719-bp-long dsDNA molecule with a GC content of 46.46%. The genome was predicted to have 436 ORFs and 7 tRNA genes. No virulence factor-related genes, antimicrobial resistance, or temperate lifestyle-associated genes were found in the phage HPP-Temi genome. Phage HPP-Temi is most closely related to the known or tentative representatives of the Pawinskivirus genus and can be proposed as a representative for the creation of a novel phage species in that genus. The phage and antibiotics (Ciprofloxacin) combination at varying phage titers (103, 106, 109) were used against P. aeruginosa Pa9 (PP334386) at 3.0 × 108 CFU/mL, which was carried out in triplicate. The result showed that combining antibiotics with phage significantly reduced the bacteria count at 103 and 106 titers, while no growth was observed at 109 PFU/mL. This suggests that the effect of phage HPP-Temi in combination with antibiotics is a potential and promising agent for the control of P. aeruginosa infections.

Prevalence and genetic characterization of linezolid resistance gene reservoirs in hospital sewage from Zhejiang Province, China

Hospital sewage represented important hotspots for the aggregation and dissemination of clinically relevant pathogens and antimicrobial resistance genes. To investigate the prevalence and molecular epidemiology of linezolid resistance genes in hospital sewage, both influent and effluent samples from 11 hospitals in Zhejiang Province, China, were collected and analyzed for linezolid resistance gene carriers. Thirty colonies of putative isolates that grew on the selective media with 10 mg/L florfenicol were randomly picked per sample. A total of 420 Gram-positive isolates, including 330 from 11 influent samples and 90 from three effluent samples, were obtained. Each isolate carried at least one of the linezolid resistance genes, including optrA, poxtA, cfr, and cfr(D), and the optrA gene was highly dominant (388/420). Enterococci displayed predominance among the linezolid resistance gene carriers in the hospital sewage, exhibiting a resistance rate to linezolid of 77.8 %. The wild-type OptrA and OptrA variants KLDP, RDK, and KLDK, all associated with high linezolid MICs, were most frequently detected. Phylogenetic analysis revealed the multispecies and polyclonal distribution of linezolid-resistant bacteria in hospital sewage, while Enterococcus faecalis sequence types (STs) 16 and 179 demonstrated the widest dissemination across different hospitals. Despite generally high genetic diversity, phylogenetic analysis showed that 87 isolates, assigned to ten STs from both sewage and other sources, were genetically related. Moreover, the genetic environment of linezolid resistance genes in isolates from sewage was similar to that from animals, humans, or the environment, with “Tn554-fexA-optrA” as the most common structure. These findings revealed the potential risk of the transmission of linezolid resistance genes through hospital sewage to other environments.

Screening of Klebsiella pneumoniae isolates reveals the spread of strong biofilm formers and class 1 integrons.

Klebsiella pneumoniae is a Gram-negative bacterium that can colonize, penetrate, and cause infections at several human anatomical locations. The emergence of hypervirulent K. pneumoniae and its ability to evade the immune system and develop antibiotic resistance has made it a key concern in the healthcare industry. The hypervirulent variants are increasingly involved in community-acquired infections. Therefore, it is pertinent to understand the biofilm formation potential among the clinical isolates. We acquired 225 isolates of K. pneumoniae from the Department of Microbiology, Symbiosis University Hospital and Research Centre (SUHRC), Pune, India over 1 year from March 2022- March 2023, and evaluated antimicrobial susceptibility, hypermucoviscous phenotype, virulence, and antimicrobial-resistant gene distribution in K. pneumoniae isolates and established a correlation between antimicrobial resistance and integrons. Most isolates were strong biofilm formers (76%). The isolates harbored one or more carbapenemase/ beta-lactamase encoding gene combinations. Hypermucoviscous (HMKP) isolates had considerably greater positive rates for iutA, magA, K2 serotype, rmpA, and rmpA2 than non-HMKP isolates. Isolates carrying integrons (43%) showed significantly more antibiotic resistance. The study reveals spread of strong biofilm formers with extensive virulence and antimicrobial-resistant genes, and integrons responsible for multi-drug resistance among the clinical isolates of K. pneumoniae in Pune, India, posing a threat to the public health and necessitating close surveillance, accurate diagnosis, control, and therapeutic management of infections.

Assessing antimicrobial resistance in pasture-based dairy farms: a 15-month surveillance study in New Zealand.

Antimicrobial resistance is a global public and animal health concern. Antimicrobial resistance genes (ARGs) have been detected in dairy farm environments globally; however, few longitudinal studies have utilized shotgun metagenomics for ARG surveillance in pasture-based systems. This 15-month study aimed to undertake a baseline survey using shotgun metagenomics to assess the relative abundance and diversity of ARGs in two pasture-based dairy farm environments in New Zealand with different management practices. There was no statistically significant difference in overall ARG relative abundance between the two dairy farms (P = 0.321) during the study period. Compared with overseas data, the relative abundance of ARG copies per 16S rRNA gene in feces (0.08-0.17), effluent (0.03-0.37), soil (0.20-0.63), and bulk tank milk (0.0-0.12) samples was low. Models comparing the presence or absence of resistance classes found in >10% of all feces, effluent, and soil samples demonstrated no statistically significant associations (P > 0.05) with "season," and only multi-metal (P = 0.020) and tetracycline (P = 0.0003) resistance were significant at the "farm" level. Effluent samples harbored the most diverse ARGs, some with a recognized public health risk, whereas soil samples had the highest ARG relative abundance but without recognized health risks. This highlights the importance of considering the genomic context and risk of ARGs in metagenomic data sets. This study suggests that antimicrobial resistance on pasture-based dairy farms is low and provides essential baseline ARG surveillance data for such farming systems.IMPORTANCEAntimicrobial resistance is a global threat to human and animal health. Despite the detection of antimicrobial resistance genes (ARGs) in dairy farm environments globally, longitudinal surveillance in pasture-based systems remains limited. This study assessed the relative abundance and diversity of ARGs in two New Zealand dairy farms with different management practices and provided important baseline ARG surveillance data on pasture-based dairy farms. The overall ARG relative abundance on these two farms was low, which provides further evidence for consumers of the safety of New Zealand's export products. Effluent samples harbored the most diverse range of ARGs, some of which were classified with a recognized risk to public health, whereas soil samples had the highest ARG relative abundance; however, the soil ARGs were not classified with a recognized public health risk. This emphasizes the need to consider genomic context and risk as well as ARG relative abundance in resistome studies.

Emergence of highly virulent and multidrug-resistant Escherichia coli in breeding sheep with pneumonia, Hainan Province, China

BackgroundSheep are a rarely raised livestock in Hainan Island, China, because of the unfavorable tropical marine climate. Here, this article reports a severe pneumonia in the sheep breeding and domestication facility caused acute mortality during the winter 2021–2022.MethodsSix sheep were clinically dissected and histopathologically observed. The bacteria were isolated and cultured by traditional methods and identified by 16S rRNA sequencing. The genotypes, serotypes, virulence genes and antimicrobial resistance genes were analyzed by PCR and whole genome sequencing. The pubMLST website was used for phylogenetic analysis of related strains. Kirby-Bauer disk diffusion method was used for antimicrobial susceptibility test. The antimicrobial susceptibility test standard was referred to the Clinical and Laboratory Standards Institute (CLSI). The virulence of bacteria was detected by mouse infection model.ResultsEtiology and histopathology examination of the pneumonia reveled pulmonary abscess and alveolar neutrophilia and pulmonary fibrinous exudates. Escherichia coli was the only bacterial species isolated, primarily from the lungs and blood of the six dead or moribund sheep, a total of 29 E. coli strains were isolated. Antimicrobial resistance profiling shows that all the isolates were resistant to six agents (penicillin, ampicillin, cephalothin, neomycin, erythromycin, and vancomycin) belonging to five classes of antibiotics, classifying them as multi drug resistant (MDR). Furthermore, genotyping analysis revealed all strains were common with 11–17 virulence factors indicating high pathogenicity. The lab mice infection model shows that all strains severely affect the health status particularly weight loss, lethargy, pneumonia and shortly lead to death. The molecular epidemiological analysis indicated most strains share the same genotype as previously reported strains in humans and other farmed animals this suggests a high possibility of cross-species transmission (CST) of virulent and MDR isolates. This CST could be from sheep to humans and other farmed animals or from humans and other farmed animals to sheep.ConclusionTherefore, this study indicates that E. coli is an emerging threat that causes sheep pneumonia in Hainan, and the quarantine of contacts is important to control the spread of virulent E. coli and the transmission of acquired resistance genes between humans and farmed animals such as sheep.

Seasonal stability of the rumen microbiome contributes to the adaptation patterns to extreme environmental conditions in grazing yak and cattle

BackgroundThe rumen microbiome plays an essential role in maintaining ruminants’ growth and performance even under extreme environmental conditions, however, which factors influence rumen microbiome stability when ruminants are reared in such habitats throughout the year is unclear. Hence, the rumen microbiome of yak (less domesticated) and cattle (domesticated) reared on the Qinghai-Tibetan Plateau through the year were assessed to evaluate temporal changes in their composition, function, and stability.ResultsRumen fermentation characteristics and pH significantly shifted across seasons in both cattle and yak, but the patterns differed between the two ruminant species. Ruminal enzyme activity varied with season, and production of xylanase and cellulase was greater in yak compared to cattle in both fall and winter. The rumen bacterial community varied with season in both yak and cattle, with higher alpha diversity and similarity (beta diversity) in yak than cattle. The diversity indices of eukaryotic community did not change with season in both ruminant species, but higher similarity was observed in yak. In addition, the similarity of rumen microbiome functional community was higher in yak than cattle across seasons. Moreover, yak rumen microbiome encoded more genes (GH2 and GH3) related to cellulose and hemicellulose degradation compared to cattle, and a new enzyme family (GH160) gene involved in oligosaccharides was uniquely detected in yak rumen. The season affected microbiome attenuation and buffering values (stability), with higher buffering value in yak rumen microbiome than cattle. Positive correlations between antimicrobial resistance gene (dfrF) and CAZyme family (GH113) and microbiome stability were identified in yak, but such relationship was negatively correlated in cattle.ConclusionsThe findings of the potential of cellulose degradation, the relationship between rumen microbial stability and the abundance of functional genes varied differently across seasons and between yak and cattle provide insight into the mechanisms that may underpin their divergent adaptation patterns to the harsh climate of the Qinghai-Tibetan Plateau. These results lay a solid foundation for developing strategies to maintain and improve rumen microbiome stability and dig out the potential candidates for manufacturing lignocellulolytic enzymes in the yak rumen to enhance ruminants’ performance under extreme environmental conditions.

Carriage and antimicrobial susceptibility of commensal Neisseria species from the human oropharynx

Commensal Neisseria (Nc) mainly occupy the oropharynx of humans and animals. These organisms do not typically cause disease; however, they can act as a reservoir for antimicrobial resistance genes that can be acquired by pathogenic Neisseria species. This study characterised the carriage and antimicrobial susceptibility profiles of Nc from the oropharynx of 50 participants. Carriage prevalence of Nc species was 86% with 66% of participants colonised with more than one isolate. Isolates were identified by MALDI-ToF and the most common species was N. subflava (61.4%). Minimum inhibitory concentrations (MICs) to penicillin, ceftriaxone, ciprofloxacin, azithromycin, tetracycline, and gentamicin were determined by agar dilution and E-test was used for cefixime. Using Ng CLSI/EUCAST guidelines, Nc resistance rates were above the WHO threshold of 5% resistance in circulating strains for changing the first line treatment empirical antimicrobial: 5% (CLSI) and 13 (EUCAST) for ceftriaxone and 29.3% for azithromycin. Whole genome sequencing of 30 Nc isolates was performed, which identified AMR genes to macrolides and tetracycline. Core gene MLST clustered Nc into three main groups. Gonococcal DNA uptake sequences were identified in two Nc clusters. This suggests that Nc have the potential AMR gene pool and transfer sequences that can result in resistance transfer to pathogenic Neisseria within the nasopharyngeal niche.

Application of a metatranscriptomics technology, CSI-Dx, for the detection of pathogens associated with prosthetic joint infections

Preoperative identification of causal organism(s) is crucial for effective prosthetic joint infection treatment. Herein, we explore the clinical application of a novel metatranscriptomic (MT) workflow, CSI-Dx, to detect pathogens associated with prosthetic joint infection. MT provides insight into transcriptionally active microbes, overcoming limitations of culture-based and available molecular methods. This study included 340 human synovial fluid specimens subjected to CSI-Dx and traditional culture-based methods. Exploratory analyses were conducted to determine sensitivity and specificity of CSI-Dx for detecting clinically-relevant taxa. Our findings provide insights into the active microbial community composition of synovial fluid from arthroplasty patients and demonstrate the potential clinical utility of CSI-Dx for aiding prosthetic joint infection diagnosis. This approach offers potential for improved sensitivity and acceptable specificity compared to synovial fluid culture, enabling detection of culturable and non-culturable microorganisms. Furthermore, CSI-Dx provides valuable information on antimicrobial resistance gene expression. While further optimization is needed, integrating metatranscriptomic technologies like CSI-Dx into routine clinical practice can revolutionize prosthetic joint infection diagnosis by offering a comprehensive and active snapshot of associated pathogens.

Survival of Antimicrobial Resistant Salmonella Heidelberg Inoculated into Microcosms of Fresh Pine Wood Shavings for Broiler Litter.

This study applied genomic characterizations to a cocktail of three Salmonella enterica serovar Heidelberg (S. Heidelberg) strains different antimicrobial resistance (AMR) profile which were inoculated into fresh pine wood shavings (PWS) microcosms. The strains were isolated from feces (SH-AAFC), carcass (SH-ARS) and thigh (SH-FSIS) of broiler chicken. SH-AAFC harbored an antimicrobial resistant gene (ARG) blaCMY-2 on an IncI1 plasmid while SH-FSIS harbored multiple ARGs (floR, cmlA1, tet(A), blaTEM-1B, ant(2'')-Ia, aph(6)-Id, aph(3'')-Ib and sul2) on an IncC plasmid. SH-ARS was pan-susceptible. The die-off of Salmonella was determined at days 0, 1, 7, 14 and 21. Antibiotic susceptibility tests and whole genome sequencing were performed on 77 isolates. At 21 days post-inoculation, Salmonella abundance decreased by 4.4 Log10 CFU/g with the water activity of PWS being correlated with Salmonella survival. SH-AAFC clonal populations survived longer in PWS than SH-FSIS and SH-ARS populations. SH-AAFC clones persisting in litter carried higher copy number of Col plasmids than their ancestors, while some SH-ARS clones acquired a lysogenic bacteriophage from SH-FSIS populations. These results suggests that mobile genetic determinants such as plasmids (which could carry ARGs) and bacteriophage plays roles in the persistence of S. Heidelberg in the PWS used as broiler litter.

Investigating antimicrobial resistance genes in probiotic products for companion animals

IntroductionOne of the greatest challenges of our time is antimicrobial resistance, which could become the leading cause of death globally within a few decades. In the context of One Health, it is in the common interest to mitigate the global spread of antimicrobial resistance by seeking alternative solutions, alongside appropriate drug selection and responsible use. Probiotics offer a potential avenue to reduce antibiotic usage; however, there is a scarcity of research that examines commercial products in terms of carrying antimicrobial resistance genes (ARGs) involved in resistance development through microbial vectors.MethodsOur study investigated 10 commercially available probiotic products for cats and dogs. Initially, we conducted phenotypic testing through determination of minimum inhibitory concentration (MIC) for antibiotics important in animal and public health. Subsequently, we performed next-generation sequencing (NGS) of the products to elucidate the genetic background behind the decrease in phenotypic sensitivity.ResultsIn total, 19 types of ARGs were identified, with 57.9% being found on plasmids, and in two cases, carriage as mobile genetic elements were found. One of the genes identified was the APH(3′)-Ia gene, capable of inactivating aminoglycoside antibiotics through phosphotransferase enzyme production regulation, while the other was the tetS gene, capable of conferring reduced sensitivity to tetracycline antibiotics through target protection.DiscussionOur findings underscore the importance of approaching antimicrobial resistance investigations from a broader perspective. We suggest that further studies in this area are justified and raise questions regarding the need to extend legally required studies on probiotic products from their use in economic livestock to their use in companion animals.

Investigation of the Antimicrobial Resistance of Important Pathogens Isolated from Poultry from 2015 to 2023 in the United States

Foodborne pathogens cause around 47.8 million illnesses in the U.S. annually, with antimicrobial misuse in food production, particularly in poultry processing, contributing significantly to this public health challenge. Misuse of antimicrobials can contribute to antimicrobial resistance (AMR) and make the treatment of pathogens increasingly difficult. This emphasizes the need to investigate antimicrobial resistance in U.S. poultry. This study analyzes data from the NCBI Pathogen Isolates Browser (2015–2023) to explore the relationships between antimicrobial-resistant pathogens, AMR genes, and antimicrobials detected with resistance in pathogens isolated from chicken and turkey. Using principal component analysis and hierarchical clustering, we mapped and profiled regional and temporal patterns of antimicrobial resistance. Salmonella enterica was the most prevalent antimicrobial-resistant pathogen across both chicken and turkey, with notable outbreaks, particularly in the Northeast. Antimicrobial-resistant Campylobacter jejuni was more prevalent in chicken, particularly in California and Georgia, while Escherichia coli and Shigella were more prominent in turkey, with concentrated antimicrobial resistance in Texas for pathogen samples isolated from chicken. Resistance to tetracycline and streptomycin was widespread, with distinct regional clusters: antimicrobial resistance was concentrated in states like Minnesota for pathogens isolated from chicken, while AMR found in pathogens isolated from turkey was more evenly distributed across the Midwest. Key AMR genes, such as tet(A), mdsA, and mdsB, also followed similar patterns, peaking in 2019 and significantly declining by 2022. The observed decline in AMR cases may be linked to improved biosecurity measures and disruptions in detection due to the COVID-19 pandemic. This comprehensive study of antimicrobial resistance in U.S. poultry provides valuable insights into resistance trends, which provide useful information to inform targeted interventions and policies to mitigate AMR threats in the poultry production industry. For consumers, these findings emphasize the importance of proper food handling and cooking practices to reduce the risk of exposure to resistant pathogens. Regulatory authorities should focus on enforcing stricter antimicrobial usage policies and enhancing surveillance systems to sustain the reduction in AMR cases.