Prevalence Of Antibiotic Resistance Genes Research Articles

Commensal bacteria in raw milk, the reservoirs for antibiotic resistance genes during cold chain transportation

The emergence and prevalence of antibiotic resistance genes (ARGs) in food pose a great threat to public health and have attracted globally attention. Shotgun metagenomics sequencing is widely used to characterize ARGs in the environment. However, the distribution, co-occurrence patterns, and host information of ARGs in raw milk to the cold chain transportation duration with shotgun metagenomics sequencing, are not fully understood in China. In this study, species annotation and ARG annotation were performed on 40 raw milk samples collected from Beijing (BJ), Hebei (HB), Inner Mongolia (NMG), Shanghai (SH), and Guangdong (GD) using shotgun sequencing based on the Illumina platform. A total of 4731 bacterial species were annotated, of which 37 were dominant (relative abundance > 1%). And also, 259 ARGs were annotated, corresponding to 69 kinds of antibiotic resistance. The NMG samples, which had the lowest bacterial community richness and bacterial diversity, were annotated with the highest number of ARGs, revealing the potential risk of bacterial resistance in NMG samples. Spearman correlation analysis and network analysis were showed that the probable bacterial hosts of multiple highly abundant ARGs (MacB and VanRC) in samples from provinces except for the BJ samples were Pseudomonas and Serratia, rather than conventionally known foodborne pathogens. It is noteworthy that the relative abundance of Pseudomonas fluorescens conformed to the Allometricl function with the cold chain transportation duration, the finding that could provide a reference for assessing the freshness and the risk assessment of antibiotic resistance genes of raw milk. In addition, the dairy industry should be aware of the risk of ARGs transferring from highly abundant commensal bacteria. Raw milk for transportation by cold chain, higher abundance of Pseudomonas in some samples should be kept in mind, and the roles of ARGs carried by these bacteria should be further investigated.

Evaluation of the presence of integrons, sul and smqnr genes and the prevalence of antibiotic resistance in Stenotrophomonas maltophilia clinical isolates

ObjectivesThe objective of this investigation was to examine the mechanisms associated with antibiotic resistance in Stenotrophomonas maltophilia clinical isolates retrieved from hospitalized patients undergoing open heart surgery in a Heart Center located in Tehran, Iran. Materials and methodsThis investigation encompassed a cross-sectional study of 60 S. maltophilia isolates, which were procured from diverse clinical specimens. Primary identification of the isolates was conducted through conventional microbiologic methods and subsequently verified by means of PCR primers. The E-test was utilized to establish the minimum inhibitory concentrations (MICs). PCR was then employed to ascertain the antibiotic resistance genes (sul1, sul2, Smqnr and intl1 - intl3). ResultsIn this study, a total of sixty clinical isolates of S. maltophilia were collected, with the majority of them being obtained from Intensive Care Units (ICU) (n = 54; 90%). The disk diffusion method yielded results indicating that 55% of the isolates were sensitive to minocycline, whereas 30% were intermediate and 15% were found to be resistant. Additionally, the MIC results revealed that the resistant rates of the isolates towards ceftazidime, cotrimoxazole and levofloxacin were 46.7%, 1.7% and 5%, respectively. The PCR amplification of three classes of integrons genes indicated that fifteen (25%) of the isolates carried int1, while no detection for intl2 and intl3 was reported. Furthermore, the prevalence of antibiotic resistance genes (sul1, sul2, and Smqnr) was identified in 15 (25%), 6 (10%), and 28 (46.7%) isolates, respectively. ConclusionThe reported increasing rate of antibiotic resistance and mobile genetic elements that could extend the resistance genes to other strains in the hospital, finally it could be an alarming issue for healthcare settings that need special attention to this strain and the epidemiological study on this issue.

Detection of Vancomycin Resistant Genes in Intrinsically Antibiotic Resistant Bacteria from the Gut Microbiota of Indonesian Individuals.

Antibiotic resistance is a global public health concern that has been exacerbated by the overuse and misuse of antibiotics, leading to the emergence of resistant bacteria. The gut microbiota, often influenced by antibiotic usage, plays a crucial role in overall health. Therefore, this study aimed to investigate the prevalence of antibiotic resistant genes in the gut microbiota of Indonesian coastal and highland populations, as well as to identify vancomycin-resistant bacteria and their resistant genes. Stool samples were collected from 22 individuals residing in Pacet, Mojokerto, and Kenjeran, Surabaya Indonesia in 2022. The read count of antibiotic resistant genes was analyzed in the collected samples, and the bacterium concentration was counted by plating on the antibiotic-containing agar plate. Vancomycin-resistant strains were further isolated, and the presence of vancomycin-resistant genes was detected using a multiplex polymerase chain reaction (PCR). The antibiotic resistant genes for tetracycline, aminoglycosides, macrolides, beta-lactams, and vancomycin were found in high frequency in all stool samples (100%) of the gut microbiota. Meanwhile, those meant for chloramphenicol and sulfonamides were found in 86% and 16% of the samples, respectively. Notably, vancomycin-resistant genes were found in 16 intrinsically resistant Gram-negative bacterial strains. Among the detected vancomycin-resistant genes, vanG was the most prevalent (27.3%), while vanA was the least prevalent (4.5%). The presence of multiple vancomycin resistance genes in intrinsically resistant Gram-negative bacterial strains demonstrated the importance of the gut microbiota as a reservoir and hub for the horizontal transfer of antibiotic resistant genes.

Characterisation of microbial communities and quantification of antibiotic resistance genes in Italian wastewater treatment plants using 16S rRNA sequencing and digital PCR

The spread of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in humans, animals and environment is a growing threat to public health. Wastewater treatment plants (WWTPs) are crucial in mitigating the risk of environmental contamination by effectively removing contaminants before discharge. However, the persistence of ARB and ARGs even after treatment is a challenge for the management of water system.To comprehensively assess antimicrobial resistance dynamics, we conducted a one-year monitoring study in three WWTPs in central Italy, both influents and effluents. We used seasonal sampling to analyze microbial communities by 16S rRNA, as well as to determine the prevalence and behaviour of major ARGs (sul1, tetA, blaTEM, blaOXA-48, blaCTX-M-1 group, blaKPC) and the class 1 Integron (int1). Predominant genera included in order: Arcobacter, Acinetobacter, Flavobacterium, Pseudarcobacter, Bacteroides, Aeromonas, Trichococcus, Cloacibacterium, Pseudomonas and Streptococcus. A higher diversity of bacterial communities was observed in the effluents compared to the influents. Within these communities, we also identified bacteria that may be associated with antibiotic resistance and pose a significant threat to human health. The mean concentrations (in gene copies per liter, gc/L) of ARGs and int1 in untreated wastewater (absolute abundance) were as follows: sul1 (4.1 × 109), tetA (5.2 × 108), blaTEM (1.1 × 108), blaOXA-48 (2.1 × 107), blaCTX-M-1 group (1.1 × 107), blaKPC (9.4 × 105), and int1 (5.5 × 109). The mean values in treated effluents showed reductions ranging from one to three log. However, after normalizing to the 16S rRNA gene (relative abundance), it was observed that in 37.5 % (42/112) of measurements, the relative abundance of ARGs increased in effluents compared to influents. Furthermore, correlations were identified between ARGs and bacterial genera including priority pathogens. This study improves our understanding of the dynamics of ARGs and provides insights to develop more effective strategies to reduce their spread, protecting public health and preserving the future efficacy of antibiotics.

Metagenomic and Culturomics Analysis of Microbial Communities within Surface Sediments and the Prevalence of Antibiotic Resistance Genes in a Pristine River: The Zaqu River in the Lancang River Source Region, China.

Microbial communities inhabiting sedimentary environments in river source regions serve as pivotal indicators of pristine river ecosystems. While the correlation between antibiotic resistome and pathogenicity with core gut bacteria in humans is well established, there exists a significant knowledge gap concerning the interaction of antibiotic resistance genes (ARGs) and human pathogenic bacteria (HPB) with specific microbes in river source basins, often referred to as "terrestrial gut". Understanding the microbial composition, including bacteria and resident genetic elements such as ARGs, HPB, Mobile Genetic Elements (MGEs), and Virulence Factors (VFs), within natural habitats against the backdrop of global change, is imperative. To address this gap, an enrichment-based culturomics complementary along with metagenomics was conducted in this study to characterize the microbial biobank and provide preliminary ecological insights into profiling the dissemination of ARGs in the Lancang River Source Basin. Based on our findings, in the main stream of the Lancang River Source Basin, 674 strains of bacteria, comprising 540 strains under anaerobic conditions and 124 under aerobic conditions, were successfully isolated. Among these, 98 species were identified as known species, while 4 were potential novel species. Of these 98 species, 30 were HPB relevant to human health. Additionally, bacA and bacitracin emerged as the most abundant ARGs and antibiotics in this river, respectively. Furthermore, the risk assessment of ARGs predominantly indicated the lowest risk rank (Rank Ⅳ) in terms of endangering human health. In summary, enrichment-based culturomics proved effective in isolating rare and unknown bacteria, particularly under anaerobic conditions. The emergence of ARGs showed limited correlation with MGEs, indicating minimal threats to human health within the main stream of the Lancang River Source Basin.

Antimicrobial Resistance and Molecular Characterization of Escherichia coli from Healthy Chickens in Shandong, China from 2009 to 2014.

Antimicrobial resistance (AMR) is a great threat to animal and public health. Here, we conducted a surveillance of Escherichia coli isolated from healthy chickens during 2009-2014 to identify the characteristics of AMR. A total of 351 (95.64%) E. coli isolates were obtained from 367 healthy chicken fecal samples collected from 6 farms located in Shandong Province, China. The susceptibility to 10 antimicrobials, the prevalence of antibiotic resistance genes (ARGs), phylogenetic clustering, and multilocus sequence typing were evaluated. The isolates exhibited high resistant rates (>95%) to ampicillin, cefotaxime, ciprofloxacin, ceftiofur, and enrofloxacin. The most prevalent ARGs were blaCTX-M (36.36%), aac(6')-Ib-cr (30.79%), qnrS (29.62%), oqxAB (27%), mcr-1 (15.83%), blaTEM (9.09%), qnrC (3.52%), qnrD (0.88%), and qepA (0.29%). Phylogenetic clustering analysis indicated that the most prevalent group was group D (37.89%), followed by group B1 (34.76%), A (24.22%), and B2 (3.13%). Fifty-seven sequence types (STs) were identified among the 124 blaCTX-M-positive strains, and the dominant STs were ST354 (13.71%), ST117 (5.65%), ST155, ST2309, and ST2505 (4.84% each). There was a significant association between 17 pairs of AMR phenotypes, 14 pairs of ARGs, and 11 pairs of AMR-ARGs. The strongest association was found between ST602 and qnrC (odds ratios: 22.2). This study implied that E. coli isolated from healthy chickens could potentially serve as a reservoir of AMR and ARGs, and significant associations exist among AMR, ARGs, phylogenetic groups, and STs. Our study highlighted the need for routine surveillance of AMR in healthy chickens, and promoting appropriate antibiotic use and implementing regular monitoring of resistance in broilers are crucial for fostering the development of the poultry industry and safeguarding public health.

Transmission of antibiotic resistance through organic amendments in arable land: A 3-year field study with pigeonpea–wheat cropping system

The worldwide emergence of antimicrobial resistance (AMR) poses a substantial risk to human health and environmental stability. In agriculture, organic amendments (derived from organic sources such as manure, and plant residues) are beneficial in restoring soil properties and providing essential nutrients to crops but raise concerns about harboring antibiotic resistance, which emphasizes the need for vigilant monitoring and strategic interventions in their application. The current study assessed the impact of farming practices (organic and conventional) in a three-year field experiment with pigeonpea-wheat cropping system, focusing on the transmission of AMR using culture-dependent and -independent approaches, and soil nutrient content. Markers for antibiotic resistance genes (ARGs) (aminoglycoside-aacA, β-lactam-blaTEM, chloramphenicol-cmlA1, macrolide-ermB, sulfonamides-sul1, sul2, and tetracycline-tetO) and integrons (intl1 and intl2) were targeted using qPCR. Manure amendments, particularly FYM1, exhibited a higher abundance of copies of ARGs compared to the rhizospheric soil. Organic farming was associated with higher copies of intl2, sul1, blaTEM, and tetO genes, while conventional farming showed increased copies of sul2 and ermB genes in the rhizosphere. Significant positive correlations were observed among soil nutrient contents, ARGs, and MGEs. The notable prevalence of ARGs linked to manure amendments serves as a cautionary note, demanding responsible management practices.

Camel Milk Resistome in Kuwait: Genotypic and Phenotypic Characterization.

Antimicrobial resistance (AMR) is one of the major global health and economic threats. There is growing concern about the emergence of AMR in food and the possibility of transmission of microorganisms possessing antibiotic resistance genes (ARGs) to the human gut microbiome. Shotgun sequencing and in vitro antimicrobial susceptibility testing were used in this study to provide a detailed characterization of the antibiotic resistance profile of bacteria and their ARGs in dromedary camel milk. Eight pooled camel milk samples, representative of multiple camels distributed in the Kuwait desert, were collected from retail stores and analyzed. The genotypic analysis showed the presence of ARGs that mediate resistance to 18 classes of antibiotics in camel milk, with the highest resistance to fluoroquinolones (12.48%) and disinfecting agents and antiseptics (9%). Furthermore, the results pointed out the possible transmission of the ARGs to other bacteria through mobile genetic elements. The in vitro antimicrobial susceptibility testing indicated that 80% of the isolates were resistant to different classes of antibiotics, with the highest resistance observed against three antibiotic classes: penicillin, tetracyclines, and carbapenems. Multidrug-resistant pathogens including Klebsiella pneumoniae, Escherichia coli, and Enterobacter hormaechei were also revealed. These findings emphasize the human health risks related to the handling and consumption of raw camel milk and highlight the necessity of improving the hygienic practices of farms and retail stores to control the prevalence of ARGs and their transmission.

Linking bacterial life strategies with the distribution pattern of antibiotic resistance genes in soil aggregates after straw addition

Straw addition markedly affects the soil aggregates and microbial community structure. However, its influence on the profile of antibiotic resistance genes (ARGs), which are likely associated with changes in bacterial life strategies, remains unclear. To clarify this issue, a soil microcosm experiment was incubated under aerobic (WS) or anaerobic (AnWS) conditions after straw addition, and metagenomic sequencing was used to characterise ARGs and bacterial communities in soil aggregates. The results showed that straw addition shifted the bacterial life strategies from K- to r-strategists in all aggregates, and the aerobic and anaerobic conditions stimulated the growth of aerobic and anaerobic r-strategist bacteria, respectively. The WS decreased the relative abundances of dominant ARGs such as QnrS5, whereas the AnWS increased their abundance. After straw addition, the macroaggregates consistently exhibited a higher number of significantly altered bacteria and ARGs than the silt+clay fractions. Network analysis revealed that the WS increased the number of aerobic r-strategist bacterial nodes and fostered more interactions between r-and K-strategist bacteria, thus promoting ARGs prevalence, whereas AnWS exhibited an opposite trend. These findings provide a new perspective for understanding the fate of ARGs and their controlling factors in soil ecosystems after straw addition. Environmental implicationsStraw soil amendment has been recommended to mitigate soil fertility degradation, improve soil structure, and ultimately increase crop yields. However, our findings highlight the importance of the elevated prevalence of ARGs associated with r-strategist bacteria in macroaggregates following the addition of organic matter, particularly fresh substrates. In addition, when assessing the environmental risk posed by ARGs in soil that receives crop straw, it is essential to account for the soil moisture content. This is because the species of r-strategist bacteria that thrive under aerobic and anaerobic conditions play a dominant role in the dissemination and accumulation of ARG.

Assessment of antibiotic resistance genes in soils polluted by chemical and technogenic ways with poly-aromatic hydrocarbons and heavy metals

Anthropogenic activities are leaving lots of chemical footprints on the soil. It alters the physiochemical characteristics of the soil thereby modifying the natural soil microbiome. The prevalence of antimicrobial-resistance microbes in polluted soil has gained attention due to its obvious public health risks. This study focused on assessing the prevalence and distribution of antibiotic-resistance genes in polluted soil ecosystems impacted by industrial enterprises in southern Russia. Metagenomic analysis was conducted on soil samples collected from polluted sites using various approaches, and the prevalence of antibiotic-resistance genes was investigated. The results revealed that efflux-encoding pump sequences were the most widely represented group of genes, while genes whose products replaced antibiotic targets were less represented. The level of soil contamination increased, and there was an increase in the total number of antibiotic-resistance genes in proteobacteria, but a decrease in actinobacteria. The study proposed an optimal mechanism for processing metagenomic data in polluted soil ecosystems, which involves mapping raw reads by the KMA method, followed by a detailed study of specific genes. The study's conclusions provide valuable insights into the prevalence and distribution of antibiotic-resistance genes in polluted soils and have been illustrated in heat maps.

Current examining methods and mathematical models of horizontal transfer of antibiotic resistance genes in the environment.

The increasing prevalence of antibiotic resistance genes (ARGs) in the environment has garnered significant attention due to their health risk to human beings. Horizontal gene transfer (HGT) is considered as an important way for ARG dissemination. There are four general routes of HGT, including conjugation, transformation, transduction and vesiduction. Selection of appropriate examining methods is crucial for comprehensively understanding characteristics and mechanisms of different HGT ways. Moreover, combined with the results obtained from different experimental methods, mathematical models could be established and serve as a powerful tool for predicting ARG transfer dynamics and frequencies. However, current reviews of HGT for ARG spread mainly focus on its influencing factors and mechanisms, overlooking the important roles of examining methods and models. This review, therefore, delineated four pathways of HGT, summarized the strengths and limitations of current examining methods, and provided a comprehensive summing-up of mathematical models pertaining to three main HGT ways of conjugation, transformation and transduction. Finally, deficiencies in current studies were discussed, and proposed the future perspectives to better understand and assess the risks of ARG dissemination through HGT.

Temporal Dynamics and Contribution of Phage Community to the Prevalence of Antibiotic Resistance Genes in a Full-Scale Sludge Anaerobic Digestion Plant.

Anaerobic digestion (AD) is an important biological resource recovery process, where microorganisms play key roles for material transformation. There has been some knowledge about the prokaryotic community and antibiotic resistance genes (ARGs) in AD, but there has been very limited knowledge of phages. In this study, samples from a full-scale AD plant were collected over 13 months, sequenced, and analyzed for viral and prokaryotic metagenomes. Totally, 3015 viral operational taxonomic units (vOTUs) were detected, mostly assigned to Caudoviricetes. The phage community had faster temporal variation than the prokaryotic community. Warm seasons harbored a higher abundance of both temperate phages and broad host-range phages. Seven ARGs of 6 subtypes were carried by 20 vOTUs, a representative ermT gene was synthesized and expressed, and the resistance activity in the host was examined, confirming the real activity of virus-carried ARGs in the AD process. Some of the ARGs were horizontally transferred between the phage and prokaryotic genomes. However, phage infection was not found to contribute to ARG transfer. This study provided an insight into the ecological patterns of the phage community, confirmed the antibiotic resistance activity of virus-carried ARGs, evaluated the contribution of phages on the ARG prevalence, and laid the foundation for the control strategies of the community and antibiotic resistance in the AD process.

Effect of a probiotic and an antibiotic on the mobilome of the porcine microbiota.

Introduction: To consider the growing health issues caused by antibiotic resistance from a "one health" perspective, the contribution of meat production needs to be addressed. While antibiotic resistance is naturally present in microbial communities, the treatment of farm animals with antibiotics causes an increase in antibiotic resistance genes (ARG) in the gut microbiome. Pigs are among the most prevalent animals in agriculture; therefore, reducing the prevalence of antibiotic-resistant bacteria in the pig gut microbiome could reduce the spread of antibiotic resistance. Probiotics are often studied as a way to modulate the microbiome and are, therefore, an interesting way to potentially decrease antibiotic resistance. Methods: To assess the efficacy of a probiotic to reduce the prevalence of ARGs in the pig microbiome, six pigs received either treatment with antibiotics (tylvalosin), probiotics (Pediococcus acidilactici MA18/5M; Biopower® PA), or a combination of both. Their faeces and ileal digesta were collected and DNA was extracted for whole genome shotgun sequencing. The reads were compared with taxonomy and ARG databases to identify the taxa and resistance genes in the samples. Results: The results showed that the ARG profiles in the faeces of the antibiotic and combination treatments were similar, and both were different from the profiles of the probiotic treatment (p < 0.05). The effects of the treatments were different in the digesta and faeces. Many macrolide resistance genes were detected in a higher proportion in the microbiome of the pigs treated with antibiotics or the combination of probiotics and antibiotics. Resistance-carrying conjugative plasmids and horizontal transfer genes were also amplified in faeces samples for the antibiotic and combined treatments. There was no effect of treatment on the short chain fatty acid content in the digesta or the faeces. Conclusion: There is no positive effect of adding probiotics to an antibiotic treatment when these treatments are administered simultaneously.

Hygiene practices and antibiotic resistance among dental and medical students: a comparative study.

Healthcare students' hand and smartphone hygiene is critical due to potential pathogenic and antibiotic-resistant bacteria transmission. This study evaluates hygiene practices in medical and dental students at Kuwait University, exploring antibiotic resistance gene prevalence. Swab samples were collected from the hands and smartphones of 32 medical and 30 dental students. These samples were cultured on Columbia Blood Agar and McConkey Agar plates to quantify bacterial colony-forming units (CFUs). The extracted DNA from these colonies underwent RT-PCR to identify antibiotic resistance genes, including tem-1, shv, blaZ, and mecA. Additionally, a questionnaire addressing hygiene practices was distributed post-sample collection. Medical students exhibited more frequent hand hygiene compared to dental students (P ≤ 0.0001). Although significantly fewer bacterial CFUs were found on medical students' smartphones (mean = 35 ± 53) than dental students' (mean = 89 ± 129) (P ≤ 0.05), no significant differences were observed in CFU counts on their hands (medical: mean = 17 ± 37; dental: mean = 96 ± 229). Detection of at least one of the targeted antibiotic resistance genes on medical (89% hands, 52% smartphones) and dental students' (79% hands, 63% smartphones) was not statistically significant. However, the prevalence of two genes, tem-1 and shv, was significantly higher on medical students' hands (78% and 65%, respectively) than on dental students' hands (32% and 28%, respectively). Clinically significant prevalence of antibiotic resistance genes were found on medical and dental students' hands and smartphones, emphasizing the importance of ongoing education regarding hand hygiene and smartphone disinfection. This continuous reinforcement in the curriculum is crucial to minimizing the risk of cross-contamination.

Deciphering the removal of antibiotics and the antibiotic resistome from typical hospital wastewater treatment systems

Hospital wastewater treatment systems (HWTSs) are a significant source and reservoir of antibiotic resistance genes (ARGs) and a crucial hub for transmitting ARGs from clinical to natural environments. However, there is a lack of research on the antibiotic resistome of clinical wastewater in HWTSs. In this study, we used metagenomics to analyze the prevalence and abundance of ARGs in five typical HWTSs. A total of 17 antibiotics from six categories were detected in the five HWTSs; β-lactam antibiotics were found at the highest concentrations, with up to 4074.08 ng·L−1. We further found a total of 21 ARG types and 1106 subtypes of ARGs with the highest percentage of multi-drug resistance genes (evgS, msbA, arlS, and baeS). The most abundant last-resort ARGs were mcr, which were detected in 100 % of the samples. HWTSs effluent is a major pathway for the transmission of last-resort ARGs into urban wastewater networks. The removal of antibiotics, antibiotic-resistant bacteria, and ARGs from HWTSs was mainly achieved by tertiary treatment, i.e., chlorine disinfection, but antibiotics and ARGs were still present in the HWTSs effluent or even increased after treatment. Moreover, antibiotics and heavy metals (especially mercury) in hospital effluents can exert selective pressure for antibiotic resistance, even at low concentrations. Qualitative analyses based on metagenome-assembled genome analysis revealed that the putative hosts of the identified ARGs are widely distributed among Pseudomonas, Acidovorax, Flavobacterium, Polaromonas, and Arcobacter. Moreover, we further assessed the clinical availability of ARGs and found that multidrug ARGs had the highest clinical relevance values. This study provides new impulses for monitoring and removing antibiotics and ARGs in the hospital sewage treatment process.

Composting reduces the risks of resistome in beef cattle manure at the transcriptional level.

Transcriptomic evidence is needed to determine whether composting is more effective than conventional stockpiling in mitigating the risk of resistome in livestock manure. The objective of this study is to compare composting and stockpiling for their effectiveness in reducing the risk of antibiotic resistance in beef cattle manure. Samples collected from the center and the surface of full-size manure stockpiling and composting piles were subject to metagenomic and metatranscriptomic analyses. While the distinctions in resistome between stockpiled and composted manure were not evident at the DNA level, the advantages of composting over stockpiling were evident at the transcriptomic level in terms of the abundance of antibiotic resistance genes (ARGs), the number of ARG subtypes, and the prevalence of high-risk ARGs (i.e., mobile ARGs associated with zoonotic pathogens). DNA and transcript contigs show that the pathogen hosts of high-risk ARGs included Escherichia coli O157:H7 and O25b:H4, Klebsiella pneumoniae, and Salmonella enterica. Although the average daily temperatures for the entire composting pile exceeded 55°C throughout the field study, more ARG and ARG transcripts were removed at the center of the composting pile than at the surface. This work demonstrates the advantage of composting over stockpiling in reducing ARG risk in active populations in beef cattle manure.IMPORTANCEProper treatment of manure before land application is essential to mitigate the spread of antibiotic resistance in the environment. Stockpiling and composting are two commonly used methods for manure treatment. However, the effectiveness of composting in reducing antibiotic resistance in manure has been debated. This work compared the ability of these two methods to reduce the risk of antibiotic resistance in beef cattle manure. Our results demonstrate that composting reduced more high-risk resistance genes at the transcriptomic level in cattle manure than conventional stockpiling. This finding not only underscores the effectiveness of composting in reducing antibiotic resistance in manure but also highlights the importance of employing RNA analyses alongside DNA analyses.

Comparison of tet(X4)-containing contigs assembled from metagenomic sequencing data with plasmid sequences of isolates from a cohort of healthy subjects.

Recently discovered tet(X) gene variants have provided new insights into microbial antibiotic resistance mechanisms and their potential consequences for public health. This study focused on detection, analysis, and characterization of Tet(X4)-positive Enterobacterales from the gut microbiota of a healthy cohort of individuals in Singapore using cultivation-dependent and cultivation-independent approaches. Twelve Tet(X4)-positive Enterobacterales strains that were previously obtained from the cohort were fully genome-sequenced and comparatively analyzed. A metagenomic sequencing (MS) data set of the same samples was mined for contigs that harbored the tet(X4) resistance gene. The sequences of tet(X4)-containing contigs and plasmids sequences were compared. The presence of the resistance genes floR and estT (previously annotated as catD) was detected in the same cassette in 10 and 12 out of the 12 tet(X4)-carrying plasmids, respectively. MS detected tet(X4)-containing contigs in 2 out of the 109 subjects, while cultivation-dependent analysis previously reported a prevalence of 10.1%. The tet(X4)-containing sequences assembled from MS data are relatively short (~14 to 33 kb) but show high similarity to the respective plasmid sequences of the isolates. Our findings show that MS can complement efforts in the surveillance of antibiotic resistance genes for clinical samples, while it has a lower sensitivity than a cultivation-based method when the target organism has a low abundance. Further optimization is required if MS is to be utilized in antibiotic resistance surveillance.IMPORTANCEThe global rise in antibiotic resistance makes it necessary to develop and apply new approaches to detect and monitor the prevalence of antibiotic resistance genes in human populations. In this regard, of particular interest are resistances against last-resort antibiotics, such as tigecycline. In this study, we show that metagenomic sequencing can help to detect high abundance of the tigecycline resistance gene tet(X4) in fecal samples from a cohort of healthy human subjects. However, cultivation-based approaches currently remain the most reliable and cost-effective method for detection of antibiotic-resistant bacteria.

Spread of antibiotic resistance genes to Antarctica by migratory birds

Recent studies have highlighted the presence of antibiotic resistance genes (ARGs) in Antarctica, which are typically indicative of human activity. However, these studies have concentrated in the Antarctic Peninsula region, and relatively less is known about ARG prevalence in East Antarctica, where human activity levels are lower compared to the Antarctic Peninsula. In addition, the mechanisms of ARG transmission to Antarctica through natural or anthropogenic pathways remain unclear. In this study, we analyzed the fecal samples of Adélie penguins and South polar skuas by using high-throughput sequencing and microfluidic quantitative PCR to detect potential pathogens and ARGs at their breeding colonies near Syowa Station in East Antarctica. These results revealed the presence of several potential pathogens in the fecal matter of both bird species. However, the HF183 marker, which indicates human fecal contamination, was absent in all samples, as well as seawater sampled near the breeding colonies. This suggests that the human fecal contamination was negligible in our study area. In addition to pathogens, we found a significant number of ARGs and metal resistance genes in the feces of both Adélie penguins and South polar skuas, with higher detection rates in skuas than in penguins. To better understand how these birds acquire and transmit these genes, we analyzed the migratory patterns of Adélie penguins and South polar skuas by geolocator tracking. We found that the skuas migrate to the tropical and subtropical regions of the Indian Ocean during the austral winter. On the other hand, Adélie penguins exhibited a more localized migration pattern, mainly staying within Antarctic waters. Because the Indian Ocean is considered one of the major reservoirs of ARGs, South polar skuas might be exposed to ARGs during their winter migration and transfer these genes to Antarctica.

Antimicrobial peptides: An alternative to antibiotic for mitigating the risks of Antibiotic resistance in aquaculture

The utilization of antibiotics increases the prevalence of antibiotic resistance genes (ARGs) in various matrices and poses the potential risk of ARG transmission, garnering global attention. Antimicrobial peptides (AMPs) represent a promising novel category of antimicrobials that may address the urgent issue of antibiotic resistance. Here, a zebrafish cultivation assay in which zebrafish were fed a diet supplemented with AMP (Cecropin A) or antibiotics was conducted to determine the effects of the intervention on the microorganisms and antibiotic resistance spectrum in zebrafish gut samples. Cecropin A treatment decreased the α-diversity of the microbiota. Moreover, NMDS (nonmetric multidimensional scaling) results revealed that the β-diversity in the microbiota was more similar between the control (CK) and Cecropin A samples than between the antibiotic treatment groups. The absolute quantity of ARGs in the AMP treatment was less than that observed in the antibiotic treatment. The findings indicated that FFCH7168, Chitinibacter and Cetobacterium were the most significant biomarkers detected in the CK, Cecropin A and antibiotic treatments, respectively. Although the use of antibiotics notably enhanced the occurrence of multidrug-resistant bacteria, the application of Cecropin A did not lead to this phenomenon. The results indicated that the application of AMPs can effectively manage and control ARGs in aquaculture.

16S rDNA-Based Amplicon Analysis Unveiled a Correlation Between the Bacterial Diversity and Antibiotic Resistance Genes of Bacteriome of Commercial Smokeless Tobacco Products.

The distribution of bacterial-derived antibiotic resistance genes (ARGs) in smokeless tobacco products is less explored and encourages understanding of the ARG profile of Indian smokeless tobacco products. Therefore, in the present investigation, ten commercial smokeless tobacco products were assessed for their bacterial diversity to understand the correlation between the inhabitant bacteria and predicted ARGs using a 16S rDNA-based metagenome analysis. Overall analysis showed the dominance of two phyla, i.e., Firmicutes (43.07%) and Proteobacteria (8.13%) among the samples, where Bacillus (9.76%), Terribacillus (8.06%), Lysinibacillus (5.8%), Alkalibacterium (5.6%), Oceanobacillus (3.52%), and Dickeya (3.1%) like genera were prevalent among these phyla. The phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt)-based analysis revealed 217 ARGs which were categorized into nine groups. Cationic antimicrobial polypeptides (CAMP, 33.8%), vancomycin (23.4%), penicillin-binding protein (13.8%), multidrug resistance MDR (10%), and β-lactam (9.3%) were among the top five contributors to ARGs. Staphylococcus, Dickeya, Bacillus, Aerococcus, and Alkalibacterium showed their strong and significant correlation (p value < 0.05) with various antibiotic resistance mechanisms. ARGs of different classes (blaTEM, blaSHV, blaCTX, tetX, vanA, aac3-II, mcr-1, intI-1, and intI2) were also successfully amplified in the metagenomes of SMT samples using their specific primers. The prevalence of ARGs in inhabitant bacteria of smokeless tobacco products suggests making steady policies to regulate the hygiene of commercial smokeless tobacco products.