An Insight into the Presence of Antimicrobial Resistance Genes in Opportunistic Pathogenic Bacteria Isolated from Farm-Reared Crickets

The detection of recombinant DNA in a vitamin B2 used as a feed additive was notified by the Belgian national authorities on 2 October 2018 via the Rapid Alert System for Food and Feed (RASFF). The European Commission requested scientific advice from EFSA on the risk posed to humans by the presence of genetically modified material in the feed additive, particularly with regard to antimicrobial resistance (AMR). EFSA assessed the analytical data from RASFF regarding the presence of AMR genes in both additive and feed. Samples of the additive and feed tested positive for the presence of DNA of a genetically modified Bacillus subtilis. The results were compatible with, but did not demonstrate the presence of, a full‐length chloramphenicol resistance gene. No information was made available on the presence of other AMR genes or viable cells of the B. subtilis. The statement provides a risk assessment pathway indicating the events needed to produce adverse human health effects from the presence of AMR genes in feed additives. Data on the likelihood of occurrence of all events are needed to produce an evidence‐based estimate of the risk. All the events are theoretically possible, but there are no scientific data available to estimate the probability of each taking place. Moreover, there is no evidence of the presence of full‐length AMR gene(s) in the vitamin B2 additive or feed; thus, it is not clear whether the first step towards AMR gene transfer is fulfilled. The sole presence of fragments of AMR genes in a feed additive is not a risk. If a full‐length AMR gene were present in a feed additive, it could lead to risks linked to its transmission to pathogens via the food chain and/or to the environmental spread of AMR bacteria/genes, potentially contributing to the environmental reservoir of AMR determinants.

The increasing prevalence of antimicrobial resistance (AMR) is a significant threat to global health. More and more multi-drug-resistant bacterial strains cause life-threatening infections and the death of thousands of people each year. Beyond disease control animals are often given antibiotics for growth promotion or increased feed efficiency, which further increase the chance of the development of multi-resistant strains. After the consumption of unprocessed animal products, these strains may meet the human bacteriota. Among the foodborne and the human populations, antimicrobial resistance genes (ARGs) may be shared by horizontal gene transfer. This study aims to test the presence of antimicrobial resistance genes in milk metagenome, investigate their genetic position and their linkage to mobile genetic elements. We have analyzed raw milk samples from public markets sold for human consumption. The milk samples contained genetic material from various bacterial species and the in-depth analysis uncovered the presence of several antimicrobial resistance genes. The samples contained complete ARGs influencing the effectiveness of acridine dye, cephalosporin, cephamycin, fluoroquinolone, penam, peptide antibiotics and tetracycline. One of the ARGs, PC1 beta-lactamase may also be a mobile element that facilitates the transfer of resistance genes to other bacteria, e.g. to the ones living in the human gut.

Whole-genome sequencing of Escherichia coli isolated from contaminated meat samples collected from the Northern Region of Ghana reveals the presence of multiple antimicrobial resistance genes.

Background Antimicrobial resistance (AMR) is a growing global public health threat. In 2019, resistant bacterial infections and respiratory tract infections were a leading cause of death in children worldwide. While the gut is a known reservoir of AMR genes, there is little data on the AMR burden in the respiratory tract. We used metagenomic RNA next-generation sequencing (mNGS) to describe bacterial AMR genes detected in the upper respiratory tract of children and compare with adults. Methods We leveraged two established cohort studies of children and adults diagnosed with acute respiratory illnesses in the inpatient and outpatient setting in California and Colorado. Nasopharyngeal (NP) swabs were collected between March to September 2020. Specimens underwent RNA extraction and paired-end Illumina sequencing. AMR genes were detected using the ARG-ANNOT database and the CZID pipeline. Children ≤18 years of age and adults≥ 40 years of age were eligible for inclusion in the analysis. We described and compared AMR genes and AMR gene classes detected in children and adults. P-values were calculated using the Wilcoxon rank-sum test. A multiple logistic regression model was fitted to evaluate the association of age cohort (children vs adults) with presence of AMR genes, while accounting for possible confounding from sex and encounter setting. 95% confidence intervals (CI) were calculated using the Wald CI. Results NP swab mNGS data from 236 patients (82 children, 154 adults) were analyzed. Children were a median age of 4 years (range: 0-17 years), and adults were a median age of 62 years (range: 40-89 years). Both cohorts were 50% male. AMR genes were detected in 44 (54%) children and 109 (71%) adults. Genes conferring resistance to beta-lactams (n=112, 47%), macrolides (n=89, 38%), and tetracyclines (n=72, 31%) were the most frequently detected. Among children, children ages 0 to 2 years had a higher number of AMR genes than children of other age groups, particularly when compared with children ages 3 to 10 years (p-value: 0.031) (Figure 1A). When comparing children with adults, children had fewer AMR genes (p-value: 0.014) as well as fewer AMR gene classes (p-value: 0.031) (Figure 1B). In the multiple logistic regression model, children still had lower odds of any AMR genes detected compared with adults (odds ratio: 0.39, 95% CI: 0.21-0.71). Figure 1. Box plots of (A) the number of antimicrobial resistance (AMR) genes detected in children stratified by age, and (B) the number of AMR genes and AMR gene classes detected in children (n=82) and adults (n=154). The size of the individual data points within the box plots correlates with the number of patients at each data point. Conclusion Children had fewer AMR genes and gene classes than adults in their upper respiratory tract microbiome. Despite this, over half of the children had detectable resistance genes, and children 2 years and younger had more AMR genes than other children. Almost half of the children had resistance genes to beta-lactams, the most frequently prescribed antibiotics to children. These findings suggest that presence of AMR organisms in the airway is common even in children, and that continued efforts to reduce AMR burden in this population are essential.

Campylobacter spp. are common pathogenic bacteria in both veterinary and human medicine. Infections caused by Campylobacter spp. are usually treated using antibiotics. However, the injudicious use of antibiotics has been proven to spearhead the emergence of antibiotic resistance. The purpose of this study was to detect the prevalence of antibiotic resistance genes in Campylobacter spp. isolated from chickens and human clinical cases in South Africa. One hundred and sixty one isolates of Campylobacter jejuni and Campylobacter coli were collected from chickens and human clinical cases and then screened for the presence of antimicrobial resistance genes. We observed a wide distribution of the tetO gene, which confers resistance to tetracycline. The gyrA genes that are responsible quinolone resistance were also detected. Finally, our study also detected the presence of the blaOXA-61, which is associated with ampicillin resistance. There was a higher (p < 0.05) prevalence of the studied antimicrobial resistance genes in chicken faeces compared with human clinical isolates. The tetO gene was the most prevalent gene detected, which was isolated at 64% and 68% from human and chicken isolates, respectively. The presence of gyrA genes was significantly (p < 0.05) associated with quinolone resistance. In conclusion, this study demonstrated the presence of gyrA (235 bp), gyrA (270 bp), blaOXA-61 and tetO antimicrobial resistance genes in C. jejuni and C. coli isolated from chickens and human clinical cases. This indicates that Campylobacter spp. have the potential of resistance to a number of antibiotic classes.

WGS is increasingly being applied to healthcare-associated vancomycin-resistant Enterococcus faecium (VREfm) outbreaks. Within-patient diversity could complicate transmission resolution if single colonies are sequenced from identified cases. Determine the impact of within-patient diversity on transmission resolution of VREfm. Fourteen colonies were collected from VREfm positive rectal screens, single colonies were collected from clinical samples and Illumina WGS was performed. Two isolates were selected for Oxford Nanopore sequencing and hybrid genome assembly to generate lineage-specific reference genomes. Mapping to closely related references was used to identify genetic variations and closely related genomes. A transmission network was inferred for the entire genome set using Phyloscanner. In total, 229 isolates from 11 patients were sequenced. Carriage of two or three sequence types was detected in 27% of patients. Presence of antimicrobial resistance genes and plasmids was variable within genomes from the same patient and sequence type. We identified two dominant sequence types (ST80 and ST1424), with two putative transmission clusters of two patients within ST80, and a single cluster of six patients within ST1424. We found transmission resolution was impaired using fewer than 14 colonies. Patients can carry multiple sequence types of VREfm, and even within related lineages the presence of mobile genetic elements and antimicrobial resistance genes can vary. VREfm within-patient diversity could be considered in future to aid accurate resolution of transmission networks.

Antimicrobial resistance is a growing threat to public health and the environment, especially in vulnerable ecosystems such as the Amazon. The confluence of the Marañón, Utcubamba, and Chinchipe rivers, known as the Pongo de Rentema, is a strategic area where water pollution could facilitate the spread of antibiotic resistance genes. This study aims to assess water quality in this region under the “One Health” approach by analyzing physicochemical parameters, heavy metals, and the presence of antimicrobial resistance genes. Water samples were collected from five sampling points during September and October 2024. Physicochemical parameters were analyzed in situ, and heavy metal concentrations were determined using atomic emission spectrophotometry. The presence of Escherichia coli and Pseudomonas aeruginosa was evaluated through selective culture, and the detection of resistance genes (marA, ermC, amp, QEP, and qEmarA) was performed using conventional PCR. Physicochemical parameters were within the limits established by Peruvian regulations, except for total dissolved solids in the Utcubamba River. Elevated levels of lead and chromium were detected at some points. Additionally, resistance genes were identified in E. coli and P. aeruginosa, providing evidence of antimicrobial resistance dissemination in the water. Water pollution in the Pongo de Rentema poses an environmental and public health risk due to the presence of heavy metals and antimicrobial resistance genes. Continuous monitoring and environmental management strategies under the “One Health” approach are recommended to mitigate these risks.

The presence and transfer of antimicrobial resistance genes from commensal bacteria in companion animals to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. The purpose of this study was to determine antimicrobial resistance gene content and the presence of genetic elements in antimicrobial resistant Escherichia coli from healthy companion animals. In our previous study, from May to August, 2007, healthy companion animals (155 dogs and 121 cats) from three veterinary clinics in the Athens, GA, USA area were sampled and multidrug-resistant E. coli (n = 36; MDR, resistance to ≥ 2 antimicrobial classes) were obtained. Of the 25 different plasmid replicon types tested by PCR, at least one plasmid replicon type was detected in 94% (34/36) of the MDR E. coli; four isolates contained as many as five different plasmid replicons. Nine replicon types (FIA, FIB, FII, I2, A/C, U, P, I1 and HI2) were identified with FIB, FII, I2 as the most common pattern. The presence of class I integrons (intI) was detected in 61% (22/36) of the isolates with eight isolates containing aminoglycoside- and/or trimethoprim-resistance genes in the variable cassette region of intI. Microarray analysis of a subset of the MDR E. coli (n = 9) identified the presence of genes conferring resistance to aminoglycosides (aac, aad, aph and strA/B), β-lactams (ampC, cmy, tem and vim), chloramphenicol (cat), sulfonamides (sulI and sulII), tetracycline [tet(A), tet(B), tet(C), tet(D) and regulator, tetR] and trimethoprim (dfrA). Antimicrobial resistance to eight antimicrobials (ampicillin, cefoxitin, ceftiofur, amoxicillin/clavulanic acid, streptomycin, gentamicin, sulfisoxazole and trimethoprim-sulfamethoxazole) and five plasmid replicons (FIA, FIB, FII, I1 and I2) were transferred via conjugation. The presence of antimicrobial resistance genes, intI and transferable plasmid replicons indicate that E. coli from companion animals may play an important role in the dissemination of antimicrobial resistance, particularly to human hosts during contact.

First report of blaOXA-24 carbapenemase gene, armA methyltransferase and aac(6′)-Ib-cr among multidrug-resistant clinical isolates of Proteus mirabilis in Algeria

Background: Joint ill is a septicemic polyarthritis condition due to the localization of pathogenic bacteria within the joints of young calves. The emergence of antibiotic resistant pathogen in such cases may lead to further complication in treatment. Thus, the present study was aimed to isolate and identify the bacterial pathogens and its antimicrobial resistant genes from joint ill cases in calves. Methods: A total of 31 pus swabs were collected from joint ill cases from calves and subjected for bacterial isolation and identification by phenotypic and genotypic method followed by determination of its antimicrobial resistant genes (tet and mecA gene) and antibiogram. Result: Based on colony characters, microscopic observation, biochemical tests, 17/31 (54.83%) Escherichia coli and 14/31 (45.16%) Staphylococcus aureus were isolated and identified phenotypically. All the isolates were further confirmed by polymerase chain reaction using E. coli and S. aureus species specific primers. The antimicrobial resistant genes carrying E. coli and S. aureus isolates were also detected, in which 4/17 (23.5%) isolates were positive for tet gene and 4/14 (28.57%) were positive for the mecA gene. The antibiotic susceptibility test showed that the isolates were highly sensitive to Enrofloxacin (100%) and Gentamicin (77%), but were resistant to Amoxyclav (70%) and tetracycline (50%). On conclusion, E. coli and S. aureus are the most common bacterial pathogens identified from this study. The presence of antimicrobial resistant genes (tet and mecA) and antibiogram pattern of the bacterial isolates indicating the possible treatment failure and serious public health risks in future.

Gelatinous zooplankton (GZ) represents an important component of marine food webs, capable of generating massive blooms with severe environmental impact. When these blooms collapse, considerable amounts of organic matter (GZ-OM) either sink to the seafloor or can be introduced into the ocean's interior, promoting bacterial growth and providing a colonizable surface for microbial interactions. We hypothesized that GZ-OM is an overlooked marine hotspot for transmitting antimicrobial resistance genes (ARGs). To test this, we first re-analyzed metagenomes from two previous studies that experimentally evolved marine microbial communities in the presence and absence of OM from Aurelia aurita and Mnemiopsis leidyi recovered from bloom events and thereafter performed additional time-resolved GZ-OM degradation experiments to improve sample size and statistical power of our analysis. We analyzed these communities for composition, ARG, and mobile genetic element (MGE) content. Communities exposed to GZ-OM displayed up to fourfold increased relative ARG and up to 10-fold increased MGE abundance per 16S rRNA gene copy compared to the controls. This pattern was consistent across ARG and MGE classes and independent of the GZ species, indicating that nutrient influx and colonizable surfaces drive these changes. Potential ARG carriers included genera containing potential pathogens raising concerns of ARG transfer to pathogenic strains. Vibrio was pinpointed as a key player associated with elevated ARGs and MGEs. Whole-genome sequencing of a Vibrio isolate revealed the genetic capability for ARG mobilization and transfer. This study establishes the first link between two emerging issues of marine coastal zones, jellyfish blooms and ARG spread, both likely increasing with future ocean change. Hence, jellyfish blooms are a quintessential "One Health" issue where decreasing environmental health directly impacts human health.IMPORTANCEJellyfish blooms are, in the context of human health, often seen as mainly problematic for oceanic bathing. Here we demonstrate that they may also play a critical role as marine environmental hotspots for the transmission of antimicrobial resistance (AMR). This study employed (re-)analyses of microcosm experiments to investigate how particulate organic matter introduced to the ocean from collapsed jellyfish blooms, specifically Aurelia aurita and Mnemiopsis leidyi, can significantly increase the presence of antimicrobial resistance genes and mobile genetic elements in marine microbial communities by up to one order of magnitude. By providing abundant nutrients and surfaces for bacterial colonization, organic matter from these blooms enhances ARG proliferation, including transfer to and mobility in potentially pathogenic bacteria like Vibrio. Understanding this connection highlights the importance of monitoring jellyfish blooms as part of marine health assessments and developing strategies to mitigate the spread of AMR in coastal ecosystems.

Antimicrobial resistance genes (ARGs) in the respiratory microbiome are poorly characterised. We compared the presence of ARGs in healthy controls with patients with chronic lung disease in a cross-sectional study, adjusted for time since antibiotic use. Bronchoalveolar lavage was collected from 100 controls, and 93 patients with chronic obstructive pulmonary disease (COPD), 13 with asthma, 34 with sarcoidosis, 12 with idiopathic pulmonary fibrosis (IPF) and 11 patients with unclassifiable interstitial lung disease (uILD). Participants had not used antibiotics 14 days prior to sampling. Shotgun metagenomic sequencing was performed with Illumina NovaSeq. ARGs were identified using the National Database of Antibiotic-Resistant Organisms. Sample reads were normalised to counts per million. In total, 38% of controls had at least one ARG, compared with 51%, 39%, 65% and 83% of patients with COPD, asthma, sarcoidosis and IPF, respectively (p=0.01). ARGs against tetracycline (33%) were the most common ARG class, followed by beta-lactam and macrolide resistance (both 26%). In a logistic regression analysis adjusted for sex, age, body composition, smoking and antibiotic use, the OR (95% CI) for having ARGs in the lower airways was 1.30 (0.70 to 2.41) in COPD, 1.00 (0.29 to 3.52) in asthma, 3.52 (1.40 to 8.83) in sarcoidosis, 6.40 (1.25 to 32.73) in IPF and 3.27 (0.76 to 14.16) in uILD compared with controls. Overall mean (SD) ARG counts per million were 403.8 (537.7) in the 35 subjects who had used antibiotics ≤3 months before bronchoscopy, compared with 197.6 (355.9) in the 228 subjects without (p=0.02). The presence of ARGs in the lower airways microbiome was significantly higher in patients with sarcoidosis and IPF than in controls. The counts per million for ARGs were significantly associated with recent antibiotic use.

Salmonella enterica serovar 4,[5],12:i:-, a monophasic variant of Salmonella Typhimurium lacking the phase 2 flagellin, is one of the common serotypes causing Salmonellosis worldwide. However, information on Salmonella serovar 4,[5],12:i:- from Guizhou Province has lacked so far. This study aimed to investigate the antimicrobial resistance, the presence of antimicrobial resistance genes and virulence genes, and characterize the MLST genotypes of Salmonella serovar 4,[5],12:i:- isolates from Guizhou province, China. We collected 363 non-typhoid Salmonella (NTS) isolates of Guizhou from 2013 to 2018. Biochemical identification, serogroups testing, and specific multiplex polymerase chain reaction (mPCR) assay were conducted to identify Salmonella 4,[5],12:i:- isolates. Isolates were determined the antimicrobial resistance by the micro broth dilution method, detected the presence of antimicrobial resistance genes and virulence genes by PCR, and examined the molecular genotyping by Multilocus sequence typing (MLST). Eighty-seven Salmonella 4,[5],12:i:- isolates were detected, accounting for 23.9% (87/363) of the total NTS isolates. All Salmonella 4,[5],12:i:- isolates showed highly resistant to sulfaoxazole (93.1%), streptomycin (90.8%), ampicillin (88.5%), tetracycline (86.2%) and doxycycline (86.2%). A high proportion (94.2%) of multi-drug resistance (MDR) isolates were found. Most (83.9%) Salmonella 4,[5],12:i:- isolates carried four antimicrobial resistance genes, especially blaTEM-1, strA-strB, sul2, and tetB genes. Salmonella 4,[5],12:i:- isolates showed a high rate of invA, sseL, mgtC, siiE, sopB, gipA, gtgB, sspH1, and sspH2 (72.4%~98.9%). On the contrary, none of the isolates were detected the spvC and pefA genes. MLST analysis revealed three sequence types (STs), and ST34 (97.7%) was the dominant sequence type. This study is the first report of Salmonella 4,[5],12:i:- in humans from Guizhou province, China. The data might be useful for rational antimicrobial usage against Salmonella 4,[5],12:i:- infections, risk management, and public health strategies in Guizhou.

Colibacillosis is a disease caused by Escherichia coli and remains a major concern in poultry production, as it leads to significant economic losses due to carcass condemnation and clinical symptoms. The development of antimicrobial resistance is a growing problem of worldwide concern. Lysogenic bacteriophages are effective vectors for acquiring and disseminating antibiotic resistance genes (ARGs). The aim of this study was to investigate the complete genome of Escherichia coli isolates from the femurs of Brazilian broiler chickens in order to investigate the presence of antimicrobial resistance genes associated with bacteriophages. Samples were collected between August and November 2021 from broiler batches from six Brazilian states. Through whole genome sequencing (WGS), data obtained were analyzed for the presence of antimicrobial resistance genes. Antimicrobial resistance genes against the aminoglycosides class were detected in 79.36% of the isolates; 74.6% had predicted sulfonamides resistance genes, 63.49% had predicted resistance genes against β-lactams, and 49.2% of the isolates had at least one of the tetracycline resistance genes. Among the detected genes, 27 have been described in previous studies and associated with bacteriophages. The findings of this study highlight the role of bacteriophages in the dissemination of ARGs in the poultry industry.

Background Antimicrobial resistance (AMR) is a global threat projected to become a leading cause of death by 2050. Persistent selective pressure from antimicrobial overuse has driven the adaptation of resistant strains. While many AMR pathways are mediated by the presence of AMR genes (ARGs), efflux pumps (EPs) represent an often overlooked contributor to multi-drug resistance (MDR). Enterobacterales such as Klebsiella pneumoniae contain chromosomal EPs and are identified as a critical threat on the WHO Bacterial Priority Pathogens List because of growing rates of MDR. Our goal is to leverage EP regulatory sequences with known ARG markers to better predict AMR from urinary tract infection (UTI) clinical isolates using next-generation sequencing. Methods We collected approximately 3000 genomic sequences with known phenotypes from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), identifying common ARGs with AMRFinder-plus. EPs and global regulators were characterized by aligning to a custom set of EP operons and global regulatory elements derived from type strains. Variant enrichment analysis was conducted to identify mutations in EP regulatory sequences that are significantly associated with resistant phenotypes. A machine learning model was developed and trained using the ARGs and enriched EP variants, then cross-validated using 369 clinical UTI isolates from our in-house strain collection. Results We examined aminoglycoside resistance in K. pneumoniae, typically mediated by ARGs such as aminoglycoside-modifying enzymes and 16S rRNA methyltransferases. A model including only ARGs predicted gentamicin resistance with 73% accuracy. Adding major efflux pumps (AcrAB-TolC, AcrD, OqxAB) and global regulators (SoxS, MarA, BaeSR) improved accuracy to 89%, suggesting a synergistic role of ARGs and efflux systems in resistance. Conclusion Incorporating EPs and regulatory sequences into AMR predictive models substantially increased sensitivity beyond ARGs alone. We identified signatures which might contribute to elevated EP capacity, and might be important genomic factors in resistance. We are continuing to adapt similar methods for additional resistance types found in K. pneumoniae, including fluoroquinolones and extended spectrum beta lactamases (ESBLs). Disclosures Gabor Fidler, PhD, Biotia Inc: Employee Mara Couto-Rodriguez, MS, Biotia: Employee Heather L. Wells, MPH, PhD Candidate, Biotia: Employee Sol Rey, BS, Biotia: Employee Tiara Rivera, B.S., Biotia: Employee John C. Papciak, BS, Biotia: Employee Ford Combs, PhD, Biotia: Employee Caitlin Otto, PhD, D(ABMM), Biotia: Employee Lorenzo Uccellini, PhD, Biotia: Employee Christopher E. Mason, PhD, Biotia: Board Member Niamh B. O'Hara, PhD, Biotia: Employee Dorottya Nagy-Szakal, MD PhD, Biotia: Employee David C. Danko, Ph.D., Biotia: Employee