Progress 09/01/23 to 08/31/24

Outputs
Target Audience: Nothing Reported Changes/Problems:Due to the pandemic, our lab was closed between March and July 2020. The pandemic has delayed the experiment-centered research. We thus requested a no-cost extension so the project can be completed further so we can investigate the mechanisms of sulfonamide bioinactivation and establish systems to mitigate their pollution in agriculture. What opportunities for training and professional development has the project provided?This project engages the training and participation of one PhD student, Chao Li, and one undergraduate student, Jason Ong. How have the results been disseminated to communities of interest?2 peer research articles were published during this fiscal year. Particularly, a paper published in Environmental Microbiology Reports presents our investigation of resistomes in multidrug-resistant Chryseobacteria. Using a combination of metagenomics-based technologies, accumulation of ARGs in biofilter systems have been investigated as biochar pores as a key regulating factor, which was published in Bioresource Technology. In addition, 10 presentations (seminar, conference platform and poster) were given at domestic and international venues and conferences to disseminate the results from this NIFA-supported project. Pham, D. N.# and M. Li* (2024). "Comparative Resistomics Analysis of Multidrug-resistant Chryseobacteria." Environmental Microbiology Reports 16(3), e13288. (DOI: 10.1111/1758-2229.13288) (Research Article) Wei, L., J. Zheng, Y. Han, X. Xu, M. Li, and L. Zhu (2024). "Insights into the roles of biochar pores toward alleviating antibiotic resistance genes accumulation in biofiltration systems." Bioresource Technology 394, 130257. (DOI: 10.1016/j.biortech.2023.130257) (Research Article) Li, M. (July 8, 2024). Molecular Interactions between Sludge Microbiomes and Emerging Contaminants: The Good, The Bad, and The Promising. Future Environment Lab, Zhejiang University. Jiayi, China. (Seminar Presentation) Li, M. (July 1, 2024). Molecular Interactions between Sludge Microbiomes and Emerging Contaminants: The Good, The Bad, and The Promising. School of the Environment, Nanjing University. Nanjing, China. (Seminar Presentation) D. Chiang, B. Chandramouli, U. Vedagiri, Y. Kunukcu, and M. Li. (June 4, 2024) Microplastics - The State of Science and Uncertainties on Risk-Based Management. 13th International Conference on Remediation of Chlorinated and Recalcitrant Compounds. Denver, CO. (Panel) Li, M. (Mar 28, 2024). Uncovering the Molecular and Microecological Basis for the Biotransformation of Sulfonamides. Division of Biology and Chemistry, Essex County College. Newark, NJ. (Seminar Presentation) D. N. Pham# and M. Li*. (June 5, 2024) Municipal Activated Sludge-Derived Microplastic Microbiomes: The Good, The Bad, and The Promising. 13th International Conference on Remediation of Chlorinated and Recalcitrant Compounds. Denver, CO. (Poster) B. Su* and M. Li. (June 3, 2024) Profiling of Target and Non-target PFAS in Agricultural Soils. 13th International Conference on Remediation of Chlorinated and Recalcitrant Compounds. Denver, CO. (Poster) C. Li* and M. Li (May 8, 2024). Enriching biofilms for effective biodegradation of commingled emerging contaminants: optimal inoculation strategy and microbial community analysis. 109th NJWEA Annual John J. (Jack) Lagrosa Conference and Exposition. Atlantic City, NJ. (Poster) C. Li* and M. Li (May 3, 2024). Enriching biofilms for effective biodegradation of commingled emerging contaminants: optimal inoculation strategy and microbial community analysis. Theobald Smith Society Spring 2024 Symposium. New Brunswick, NJ. (Talk) M. Li* and D. N. Pham (May 3, 2024). Uncovering the molecular and microecological basis for the biotransformation of sulfonamides. Theobald Smith Society Spring 2024 Symposium. New Brunswick, NJ. (Poster) M. Li* (Apr 10, 2024). Uncovering the Molecular and Microecological Basis for the Biotransformation of Antimicrobials by Rhizobacteria and Endophyte. Soil Health Project Director Meeting, National Institute of Food and Agriculture (NIFA). Kansas City, MO. (Talk) What do you plan to do during the next reporting period to accomplish the goals?(1) We plan to compare the protein structures of SA-degrading enzymes in RD1, LD2, and other species to investigate the key residues that govern the molecular interactions for SA catalysis. (2) We will further investigate factors that can affect the antimicrobial biotransformation in biofilm-based filtration systems and assess the key degraders and mechanisms responsible for the degradation processes.

Impacts
What was accomplished under these goals? (1) To validate the HGT potential of the sadA gene/cluster from Lysobacter sp. RD1, mating experiments were employed with three individual sludge microbiomes (L, P, and R). Under the selection pressure of 50 mg/L SMX for 16 h, sadA from RD1 was successfully transferred to all three sludge microbiomes, as indicated by sadA HGT indices ranging from 3.6 and 12.3. Indices exceeding 1 in the post-mating sludge consortia confirmed the presence of sadA in sludge microorganisms in addition to RD1 cells lingering in the mixture. In contrast, sadA remained below the DL (i.e., 400 copies) in sludge samples without RD1 mating, regardless of SMX exposure. These results clearly demonstrate the transfer of sadA from RD1 to sludge microbiomes under high selective pressure of SMX. At lower SMX concentration of 100 µg/L, sadA HGT indices were around and slightly below 1. This underscores future investigations of sadA HGT at similar or lower SMX concentrations, but over longer mating time. (2) The three post-mating consortia were further examined to determine their ability to degrade SMX. After 5 days of incubation, the highest SMX biodegradation was observed in the post-mating consortium L_RD1 (from the mating between Sludge L and RD1), where 38.9±0.0 mg/L SMX was reduced to 17.3±16.0 mg/L (p < 0.05). This L_RD1 consortium was selected for a dilution-to-extinction experiment to identify potential recipients of sadA from RD1. As the dilution increased from 10-5 to 10-7, SMX removal efficiency gradually decreased from nearly 100% to 57.9%, reflecting the reduction of SMX degraders along the dilution gradient. Further dilution to 10-8 resulted in the complete loss of SMX biodegradation activity, indicating the extinction of sadA-carrying bacteria. In contrast, complete removal of SMX was achieved in the RD1 pure culture, irrespective of dilution levels. Negative controls containing only sludge L at varying dilutions showed no significant SMX degradation, confirming the absence of sadA or SMX degraders in the original sludge microbiome. Microbial community analysis of the serially diluted L_RD1 consortium revealed a decrease in total ASVs along the dilution gradient, from 158 ASVs in the 10-5 dilution to 118 ASVs in the 10-8 dilution, indicating the reduced microbial diversity. Notably, no ASVs closely related to Lysobacter sp. RD1 were detected across the 10-5 to 10-8 dilutions, indicating (1) indigenous sludge bacteria in Sludge L outcompeted this amended strain and (2) the observed SMX degradation was due to sludge bacteria that have received the sadA gene from RD1. Six ASVs, identified as Rhizobium species, Comamonas terrigena, Raoultella ornithinolytica, and Klebsiella species, were absent in the 10-8 dilution, where SMX biodegradation ceased. These ASVs also exhibited declining relative abundances along the dilution gradient, with enrichment indices decreasing by 1.1-4.7-fold across 10-5, 10-6, and 10-7 dilutions. These results suggest that these six ASVs were possible recipients of the sadA gene/cluster from RD1. It is important to note that all six ASVs are Gram-negative Pseudomonadota. Members of Raoultella ornithinolytica and Klebsiella species are known human pathogens associated with multidrug resistance and severe health effects spanning from respiratory infection to bacteremia. Further efforts involved with advanced biomolecular tools (e.g., epic PCR and chromatin conformation capture sequencing) or mating between RD1 and these identified bacteria are underscored to conclusively identify the recipients of sadA and unravel the associated horizontal transfer mechanisms. (3) Chryseobacteria consists of important human pathogens that can cause a myriad of nosocomial infections. We isolated four multidrug-resistant Chryseobacterium bacteria from activated sludge collected at domestic wastewater treatment facilities in the New York Metropolitan area. Their genomes were sequenced with Nanopore technology and used for a comprehensive resistomics comparison with 211 Chryseobacterium genomes available in the public databases. A majority of Chryseobacteria harbor 3 or more antibiotic resistance genes (ARGs) with the potential to confer resistance to at least two types of commonly prescribed antimicrobials. The most abundant ARGs, including β-lactam class A (blaCGA-1 and blaCIA) and class B (blaCGB-1 and blaIND) and aminoglycoside (ranA and ranB), are considered potentially intrinsic in Chryseobacteria. Notably, we reported a new resistance cluster consisting of a chloramphenicol acetyltransferase gene catB11, a tetracycline resistance gene tetX, and two mobile genetic elements (MGEs), IS91 family transposase and XerD recombinase. Both catB11 and tetX are statistically enriched in clinical isolates as compared to those with environmental origins. In addition, two other ARGs encoding aminoglycoside adenylyltransferase (aadS) and the small multidrug resistance pump (abeS), respectively, are found co-located with MGEs encoding recombinases (e.g., RecA and XerD) or transposases, suggesting their high transmissibility among Chryseobacteria and across the Bacteroidota phylum, particularly those with high pathogenicity. High resistance to different classes of β-lactam, as well as other commonly used antimicrobials (i.e., kanamycin, gentamicin, and chloramphenicol), was confirmed and assessed using our isolates to determine their minimum inhibitory concentrations. Collectively, though the majority of ARGs in Chryseobacteria are intrinsic, the discovery of a new resistance cluster and the co-existence of several ARGs and MGEs corroborate interspecies and intergenera transfer, which may accelerate their dissemination in clinical environments and complicate efforts to combat bacterial infections. (4) We investigated the effects of biochar pores on ARG emergence and related microbial response mechanisms in bench-scale biofiltration systems. Results showed that biochar pores effectively reduced the absolute copies of the corresponding ARGs sul1 and sul2 by 54.1% by lowering the sorbed-SMX's bioavailability compared to non-porous anthracite. An investigation of antimicrobial resistomes revealed a considerable decrease in the abundance and diversity of ARGs and mobile gene elements. Metagenomic and metaproteomic analysis demonstrated that biochar pores induced the changeover of microbial defense strategy against SMX from blocking SMX uptake by EPS absorbing to SMX biotransformation. Microbial SOS response, antibiotic efflux pump, EPS secretion, and biofilm formation were decreased. Functions related to SMX biotransformation, such as sadABC-mediated transformation, xenobiotics degradation, and metabolism, were significantly promoted.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Pham, D. N.# and M. Li* (2024). "Comparative Resistomics Analysis of Multidrug-resistant Chryseobacteria." Environmental Microbiology Reports 16(3), e13288. (DOI: 10.1111/1758-2229.13288)

Progress 09/01/22 to 08/31/23

Outputs
Target Audience: Nothing Reported Changes/Problems:Due to the pandemic, our lab was closed between March and July 2020. The pandemic has delayed the experiment-centered research. We thus requested a no-cost extension so the project can be completed further so we can investigate the mechanisms of sulfonamide bioinactivation and establish systems to mitigate their pollution in agriculture. What opportunities for training and professional development has the project provided?This project engages the training and participation of one PhD student, Dung Ngoc Pham, and one undergraduate student, Jason Ong. How have the results been disseminated to communities of interest?2 peer research articles were published during this fiscal year. Particularly, a paper published in Archives of Microbiology presents our investigation of ARGs in maize rhizosphere in the US as compared those in Europe and South Africa. Using a similar metagenomics-based approach, diazotrophic root endophytes were characterized in Chinese silvergrass, which was published in Microbiome. In addition, 13 presentations (seminar, conference platform and poster) were given at domestic and international venues and conferences to disseminate the results from this NIFA-supported project. Pham, D.#, Q. Wu#, and M. Li* (2023). "Global profiling of antibiotic resistomes in maize rhizospheres." Archives of Microbiology 205 (3), 89. (DOI: 10.1007/s00203-023-03424-z) (Research Article) Li, Y., R. Yang, M. M. Ha?ggblom, M. Li, L. Guo, B. Li, M. Kolton, Z. Cao, M. Solemani, Z. Chen, Z. Xu, W. Gao, B. Yan, and W. Sun (2022). "Characterization of diazotrophic root endophytes in Chinese silvergrass (Miscanthus sinensis)." Microbiome 10 (1), 1-12. (DOI: 10.1186/s40168-022-01379-9) (Research Article) Li, M. (Feb 28, 2023). How Microbes Enable the Elimination of "Forever" Contaminants? Department of Environmental Health and Engineering, Johns Hopkins University. Baltimore, MD. (Seminar Presentation) Li, M. (Feb 8, 2023) Combating the Climate Crisis via Sustainable Water Management in Rice Agriculture. Dhauli Webinar Series. Odisha, India. (Virtual Webinar Presentation) Li, M. (Nov 4, 2022). How Microbes Enable the Elimination of "Forever" Contaminants? Department of Civil and Environmental Engineering, Princeton University. Princeton, NJ. (Seminar Presentation) Pham, D. N.# and M. Li*. (Jun 7, 2023) Multidrug Resistant Chryseobacteria and Their Health Implications. WATERMICRO23 21st Symposium on Health-related Water Microbiology. Darwin, Australia. (Poster) Pham, D. N.# and M. Li*. (Jun 5, 2023) Microplastics as Hubs Enriching Antibiotic-Resistant Bacteria and Pathogens in Municipal Activated Sludge. WATERMICRO23 21st Symposium on Health-related Water Microbiology. Darwin, Australia. (Poster) S. Zhang, D. N. Pham#, C. Li#, L. Axe, and M. Li*. (Jun 1, 2023) Effective Removal of Water Contaminants of Emerging Concern by Biologically Active Filters. IWA LET2023 Conference. Daegu, South Korea. (Talk) D. N. Pham# and M. Li*. (May 10, 2023) Municipal Activated Sludge-Derived Microplastic Microbiomes: The Good, The Bad, and The Promising. 2023 Bioremediation Symposium. Austin, TX. (Talk) D. N. Pham# and M. Li*. (May 10, 2023) Discovery of Gram-Negative Sulfonamide Degraders from Municipal Activated Sludge. 2023 Bioremediation Symposium. Austin, TX. (Poster) Pham, D. N.# and M. Li*. (Nov 30, 2022) Multidrug Resistant Chryseobacteria and Their Health Implications. PASTEUR2022. Warsaw, Poland. (e-Poster) Pham, D. N.# and M. Li*. (Nov 30, 2022) Unveiling the Microbiomes in Plastisphere Derived from Microplastics at Municipal Wastewater Treatment Settings. PASTEUR2022. Warsaw, Poland. (Poster) Pham, D. N.# and M. Li*. (Nov 30, 2022) Sulfonamide Resistance via Antibiotic Inactivation and Transmission among Gram-negative Bacteria in Municipal Activated Sludge. PASTEUR2022. Warsaw, Poland. (Poster) M. Li*. (Oct 28, 2022) Combating the Climate Crisis via Biochar-Enabled Sustainable Water Management in Rice Agriculture. Regional Symposium on Climate Change, Planetary, and Human Health: Challenges and Opportunities. New Brunswick, NJ. (Talk) Pham, D. N.#, L. Clark#, and M. Li*. (Sept 13, 2022) Microplastics as Hubs Enriching Antibiotic-resistant Bacteria and Pathogens in Municipal Activated Sludge. IWA World Water Congress 2022. Copenhagen, Denmark. (Talk) What do you plan to do during the next reporting period to accomplish the goals?(1) We plan to conduct liquid mating between the consortium RD1 that carries sadA and different activated sludge communities, which can help us understand the potential transfer of the sadA cassette between distantly related microorganisms. (2) We will further establish biofilm-enabled filtration systems and valuate their potential to mitigate antimicrobials and prevent the dissemination of antibiotic resistance in agricultural environment.

Impacts
What was accomplished under these goals? (1) We further sequenced the complete genomes of the two isolated sulfonamides-degrading Pseudomonadota from municipal activated sludge and investigated the mobility potentials of sadA and associated genes uncovered in their genomes. Oxford Nanopore Technology (ONT) was employed for the whole genome sequencing of the isolates given its capability of generating long sequencing reads (30 to 50-kb reads on average). This extended read length enables the identification of repetitive sequences, critical for the assembly of resistance clusters, particularly those acquired via horizontal gene transfer (HGT), and facilitates the recovery of intact chromosomes and plasmids. The genomes of both strains RD1 and LD2 contain sadA genes that are nearly identical (i.e., 99.6% nucleotide identity), sharing 97.7% nucleotide similarity with that of sadA from Microbacterium sp. BR1. Further phylogenetic analysis revealed that both SadAs in these two Gram-negative isolates showed closer relations to those in Microbacteriaceae than other three types of SadAs reported in Micrococcaceae. Members of Lysobacter occur naturally in soil and water and are well known for their capability of degrading various organic toxins and contaminants, for example ochratoxin A and naphthalene. A recent study by Chen et al. indicated the involvement of Lysobacter in the biodegradation of both SMX and SDZ given its dominance in SMX-degrading consortia that carried sadA genes. Similarly, Xanthobacter species were dominant in aerobic/anaerobic sludges and electrochemical reactors contaminated with SMX in a range of 1~3 mg/L. A mining of sadA genes in NCBI-NR and IMG-NR databases revealed the presence of sadA in Gram-positive bacteria of the phylum Actinomycetota but not in Gram-negative bacteria. (2) Sequence alignment of plasmid PRD1 from Lysobacter sp. RD1, plasmid PLD24 from Xanthobacter sp. LD2, and genomes of eight known SA-degrading Actinomycetota revealed a possible recent transfer of sadA genes. SadA genes detected in RD1 and LD2 shared >97.0% nucleotide similarity to those in Microbacterium sp. BR1 and other SA-degrading Actinomycetota. The sadA clusters of Lysobacter sp. RD1 and Xanthobacter sp. LD2 were on plasmids in contrast to those found in most Actinomycetota on the chromosome. Notably, a cluster of merA-merR-SRAP-sadA-yceI was identified on both sadA-containing plasmids PRD1 and PLD24 of these two Gram-negative SA degraders. This cluster was also found on the putative plasmid of the SA-degrading Candidatus Leucobacter sulfonamidivorax isolate GP.31 In addition to sadA in this cluster, there are two genes associated with cell stress and two genes that confer mercury resistance. YceI homologs that are associated with adaptation to stresses (e.g., methyl gallate and acidity) were also found in the flanking regions of sadA genes on all four Microbacterium spp. chromosomes that are known with SA degradation abilities. A recent study on genome comparison of sadA-carrying Actinomycetota species proposed the involvement of yceI in increasing uptake of SA, which would subsequently support the degradation of SAs by SadA.31 Unlike yceI, mercury (II) reductase gene merA and metal-responsive gene-regulator merR are located in separate contigs from sadA on the chromosomes of three Microbacterium spp. and one Arthrobacter. MerR and merA are involved in the detoxification of mercuric ion Hg2+ by reducing it to elemental mercury Hg0. These mercury resistance genes, part of the mer operon, are often located on plasmids or transposons such as Tn5053 and Tn21, which also frequently carry antibiotic resistance genes (ARGs). The co-localization of mercury resistance genes and sadA in RD1 and LD2 may enhance bacterial survival and promote genetic exchange, particularly at WWTPs where heavy metals and antibiotics commingle. Further, SOS response-associated peptidase (SRAP) is known for its role in a transcriptional regulatory mechanism to addresses DNA damage and regulates plasmid copy numbers. Co-localization of sadA with these heavy metal resistance and stress responsive genes may be due to co-selection during bacterial evolution and formation of such a resistance cluster may promote mobility among hosts. Some of these genes (e.g., yceI) may be involved in SA transfer and inactivation, though further molecular investigation is warranted to characterize their functions. Surrounding this merA-merR-SRAP-sadA-yceI cluster, a maximum span of approximately 13.4 kbp encompassing coding and non-coding regions of the PRD1 plasmid, were >99.0% identical to regions detected on chromosomes of Microbacterium sp. BR1, Microbacterium sp. CJ77, Microbacterium sp. C448, and Candidatus Leucobacter sulfonamidivorax isolate GP. This finding suggested a recent transfer of sadA across these Gram-positive and Gram-negative bacteria, which has been considered a major ARG transfer barrier.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2023 Citation: Wei, L., J. Zheng, Y. Han, X. Xu, M. Li, and L. Zhu (2024). "Insights into the roles of biochar pores toward alleviating antibiotic resistance genes accumulation in biofiltration systems." Bioresource Technology 394, 130257.

Progress 09/01/21 to 08/31/22

Outputs
Target Audience: Nothing Reported Changes/Problems:Due to the pandemic, our lab was closed between March and July 2020. The pandemic has delayed experiment-centered research. We thus requested a no-cost extension so the project can be completed further so we can investigate the mechanisms of sulfonamide bioinactivation and establish systems to mitigate their pollution in agriculture. What opportunities for training and professional development has the project provided?This project engages the training and participation of one postdoc, Dr. Qiong Wu, one PhD student, Dung Ngoc Pham, and one undergraduate student, Jason Ong. How have the results been disseminated to communities of interest?8 presentations (seminar, conference platform and poster) were given at domestic and international venues and conferences to disseminate the results from this NIFA-supported project. Li, M. (Feb 3, 2022). Water Microbiomes: The Good, The Bad, and The Promising. Department of Civil and Environmental Engineering, Northeastern University. Boston, MA. (Seminar Presentation) Pham, D. N.#, L. Clark#, and M. Li*. (Aug 15, 2022) Microplastics as Hubs Enriching Antibiotic-resistant Bacteria and Pathogens in Municipal Activated Sludge. ISME18. Lausanne, Switzerland. (Poster) Pham, D. N.*, L. Clark#, and M. Li. (Jun 30, 2022) Microplastics as Hubs Enriching Antibiotic-resistant Bacteria and Pathogens in Municipal Activated Sludge. 2022 AEESP Conference. St. Louis, MO. (Poster) Pham, D. N.*, Q. Wu#, and M. Li. (Jun 10, 2022) Multidrug Resistant Chryseobacteria and Their Health Implications. ASM Microbe 2022. Washington, DC. (Poster) Pham, D. N.* and M. Li. (Jun 10, 2022) Discovery of Gram-Negative Sulfonamide Degraders from Municipal Activated Sludge. ASM Microbe 2022. Washington, DC. (Poster) Pham, D. N.#, L. Clark#, and M. Li*. (Jun 10, 2022) Microplastics as Hubs Enriching Antibiotic-resistant Bacteria and Pathogens in Municipal Activated Sludge. ASM Microbe 2022. Washington, DC. (Plenary Talk) Pham, D. N.#, L. Clark#, and M. Li*. (Jun 3, 2022) Microplastics as Hubs Enriching Antibiotic-resistant Bacteria and Pathogens in Municipal Activated Sludge. ACS MARM 2022. Ewing, NJ. (Talk) Pham, D. N.#, L. Clark#, and M. Li*. (May 25, 2022) Microplastics as Hubs Enriching Antibiotic-resistant Bacteria and Pathogens in Municipal Activated Sludge. 2022 Chlorinated Conference. Palm Springs, CA. (Poster) What do you plan to do during the next reporting period to accomplish the goals?(1) We plan to conduct liquid mating between the consortium RD1 that carries sadA and different microbial communities, which can help us understand the potential transfer of the sadA cassette between distantly related microorganisms. (2) We will further establish biofilm-enabled filtration systems and valuate their potential to mitigate antimicrobials and prevent the dissemination of antibiotic resistance in agricultural environment.

Impacts
What was accomplished under these goals? (1) We conducted a survey of resistomes in maize rhizosphere from Michigan, California, the Netherlands, and South Africa, and investigated potential associations with host bacteria and soil management practices in the crop field. RbpA, vanRO, mtrA, and dfrB were prevalently found across most studied regions, implying their intrinsic origins. Further analysis revealed that RbpA, vanRO, and mtrA are mainly harbored by native Actinobacteria with low mobility since mobile genetic elements were rarely found in their flanking regions. (2) Notably, a group of dfrB genes are adjacent to the recombination binding sites (attC), which together constitute mobile gene cassettes, promoting the transmission from soil bacteria to human pathogens. DfrB was often detected in a broad spectrum of hosts within the phylum of Proteobacteria (e.g., Escherichia, Pseudomonas, and Staphylococcus) in human and animal samples, wastewater samples, and field soil samples regardless of manure amendment. Therefore, it is likely that dfrB genes from maize rhizosphere are highly mobile and transmissible as promoted by the adjacent MGEs. They can be of health concern considering the possibility of transferring the dfrB genes from maize rhizosphere to human pathogens through food production and consumption. (3) We also assembled 159 metagenome assembled genomes (MAGs) recovered from Michigan maize rhizosphere metagenomes, and 9 drug-resistant MAGs were identified. Among them, MAG 3300013105_29 and 3300026116_28 annotated as Klebsiella planticola and Enterobacter sp., respectively, contain multiple ARGs. In particular, the MAG of Klebsiella planticola harbors 7 ARGs, conferring resistance to elfamycin (EF-Tu), fosfomycin (Ptsl), triclosan (fabI), and multidrug (CRP, H-NS, marA, and soxS). The Enterobacter MAG carries 15 ARGs encoding resistance to beta-lactam (blaACT-6), fluoroquinolone (parE), fosfomycin (FosA2, ptsI, and UhpA), triclosan (fabI and gyrA), and multidrug (acrA, cpxA, CRP, emrR, HN-S, marA, oqxB, and ramA). It is known that many Enterobacter species, such as E. cloacae and E. asburiae species, are multidrug-resistant and responsible for causing nosocomial infections, spanning from respiratory infections to bacteremia. In this regard, we screened the presence of ARGs in the genome database of E. cloacae and E. asburiae that were isolated from soils and plants. All eight genomes of E. cloacae and E. asburiae available on JGI revealed the presence of four ARGs, including blaACT for beta-lactam, rpoB for rifamycin, acrB for multidrug, and parC for fluoroquinolone. Note that acrA and acrB, a component of acrA-acrB-TolC multidrug efflux system, were also identified in the Enterobacter MAG recovered from Michigan rhizosphere metagenomes. Moreover, Enterobacter species are known to contribute significantly to the dissemination of ARGs, such as blaTEM, blaCTX, and blaKPC genes. Hence, extra attention is needed to be drawn to Enterobacter and other species that carry multiple ARGs, since they can adapt to the selection pressure of antibiotics and promote the acquisition and dissemination of ARGs in the maize rhizosphere. (4) The network analysis revealed co-occurrence patterns of ARGs in Michigan maize rhizosphere metagenomes. Two ARG clusters I and II were distinctively formed. The multidrug resistance genes (acrA, adeR, cpxA, CRP, H-NS, marA, and ramA), which accounted for 81.6% of the total ARGs in Cluster I, were connected with beta-lactam (blaACT), elfamycin (EF-Tu), fluoroquinolone (emrR and parE), fosfomycin (FosA2, PtsI, and UhpA), and/or triclosan (fabI) resistance genes. The other cluster (II) consisted of a connection of only the beta-lactam resistance gene (blaOXA) and the multidrug resistance gene (soxS). Interestingly, in both clusters, ARGs from different ARG types co-existed more commonly than those from the same ARG types. (5) Overall, we investigated a broad spectrum of antibiotic resistance in maize rhizosphere from 4 regions in 3 continents across the world, including Michigan, California, South Africa and the Netherlands. Regions like South Africa and the Netherlands showed low detection of a similar collection of ARGs, which are mostly intrinsic based on our investigation. In contrast, Michigan samples exhibited a much higher diversity and abundance of ARGs, many of which were of human/animal origins and/or located adjacent to MGEs. These concerning ARGs may be associated with the long history of manure fertilization, despite that many factors (e.g., meteorology and soil properties) and agricultural practices can affect the resistomes in maize rhizosphere. The presence of these resistance determinants can increase the likelihood of their entry into the crop products and subsequently promote the spreading of their resistance genes to human pathogens through direct contact with the agricultural soils or consumption of contaminated maize products. These results suggest that maize rhizosphere resistomes can be distinctive and affected by many factors, particularly those relevant to agricultural practices. With the increasing availability of relevant metagenomes in maize rhizosphere through variable-controlled environments and intercontinental sampling, more conclusive correlations can be drawn between ARG profiling and agricultural practices and other impacting factors. (6) We also investigated the roles of diazotrophic endophytes in promoting the growth of pioneer plant Chinese silvergrass (Miscanthus sinensis) in mine tailings. Using a combination of field sampling, DNA-stable isotope probing (SIP) analysis, and pot experiments, bacteria belonging to the genera Herbaspirillum, Rhizobium, Devosia, Pseudomonas, Microbacterium, and Delftia are crucial endophytes. Further, DNA-SIP using 15N2 identified Pseudomonas, Rhizobium, and Exiguobacterium as putative diazotrophic endophytes of M. sinensis. Metagenomic-binning suggested that these bacteria contained essential genes for nitrogen fixation and plant growth promotion. Finally, two diazotrophic endophytes Rhizobium sp. G-14 and Pseudomonas sp. Y-5 were isolated from M. sinensis. Inoculation of another pioneer plant in mine tailings, Bidens pilosa, with diazotrophic endophytes resulted in successful plant colonization, significantly increased nitrogen fixation activity, and promotion of plant growth, suggesting their implications for phytoremediation.

Publications

• Type: Journal Articles Status: Published Year Published: 2022 Citation: Li, Y., Yang, R., H�ggblom, M.M. et al. Characterization of diazotrophic root endophytes in Chinese silvergrass (Miscanthus sinensis). Microbiome 10, 186 (2022). https://doi.org/10.1186/s40168-022-01379-9
• Type: Journal Articles Status: Published Year Published: 2023 Citation: Pham, D.N., Wu, Q. & Li, M. Global profiling of antibiotic resistomes in maize rhizospheres. Arch Microbiol 205, 89 (2023). https://doi.org/10.1007/s00203-023-03424-z

Progress 09/01/20 to 08/31/21

Outputs
Target Audience: Nothing Reported Changes/Problems:Over the pandemic, more progress has been achieved from metagenomic analyses that rely on computation efforts. This helps us to design and plan laboratory experiments to validate findings unveiled by metagenomics studies. What opportunities for training and professional development has the project provided?This project engages the training and participation of one postdoc, Dr. Qiong Wu, and one PhD student, Dung Ngoc Pham. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?(1) We plan to conduct liquid mating between the consortium RD1 that carries sadA and different microbial communities, which can help us understand the potential transfer of the sadA cassette between distantly related microorganisms. (2) We will further investigate the sources and hosts of ARGs dominant in maize rhizosphere and explore the implications in agriculture management.

Impacts
What was accomplished under these goals? (1) We successfully obtained a gram-negative bacterium, Xanthobacter sp. LD2, and a consortium RD1, which both carry the sulfonamide monooxygenase gene sadA through the enrichment of activated sludge samples. The whole-genome sequencing of these gram-negative isolate/consortium was carried out using the third generation sequencing approach, Nanopore. In their genome/metagenome, we identified sadA genes with high similarities to those previously reported in gram-positive Micrococcaceae. The identification of sadA genes in a transposon-rich region, potentially located in plasmids harbored by our isolates and consortia, suggested sadA can be transferred beyond the boundaries of gram-positive bacteria and represent a novel mechanism for antibiotic resistance transmission in aquatic biota. (2) In the 52 metagenomes of maize rhizosphere, ARG-carrying contigs (ACCs) were screened by searching against the ResFinder database22 and the Comprehensive Antibiotic Resistance Database (CARD)23 using Blastn with an identity of ≥70% and a query coverage of ≥60% within the Pan Resistome Analysis Pipeline (PRAP).24 Redundant ARGs were manually removed from the combined data set. Then, sequences of ARGs were extracted from ACCs using the seqtk toolkit. Across four studied regions, a total of 653 ACCs were detected in 38 out of 52 maize rhizosphere metagenomes. The total ARG abundance varied greatly from 0.09~4.50 rpm, and the detected ARGs can be categorized in to 18 types and 68 subtypes. Michigan maize rhizosphere metagenomes showed the greatest abundance and diversity of ARGs. Despite the differences in soil properties and environmental characteristics, the high abundance and diversity of resistomes in Michigan samples were likely attributed by the usage of manure from antibiotic-treated animals and the long growth time. (3) Notably, blaOXA (an averaged relative abundance of 0.21 rpm, n = 9), blaACT (0.19 rpm, n = 6), and blaTEM (0.20 rpm, n = 3) were the top three abundant ARGs, together accounting for approximately 30% of the total ARGs in Michigan maize rhizosphere metagenomes on average. These ARGs encode beta-lactamases that can inactivate a broad spectrum of commonly used beta-lactams, particularly those in cephalosporin and penam classes, by hydrolyzing the β-lactam ring. The dominance of these β-lactam resistance genes in the maize rhizosphere are concerning as they may pose serious threats to public health.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: T�ncsics, Andr�s, Mengyan Li, and ?ukasz Chrzanowski. "New Insights Into the Biodegradation of Organic Contaminants in Subsurface Ecosystems: Approaches and Achievements of the Multiomics Era." Frontiers in Microbiology 12 (2021): 946.

Progress 09/01/19 to 08/31/20

Outputs
Target Audience:In addition to the scientific communities in the fields of antibiotic resistance and microbial ecology, outcomes of this project will benefit a broader range of communities, including (1) corn farmers with concerns of antibiotic and antibiotic resistance contamination, (2) agricultural and environmental agencies that promote green agriculture, (3) manufacturers of downstream corn products, such as corn starch and syrup, (4) livestock farmers who intend to enhance the feed quality and product quality and yield, (5) pharmaceutical industries with liability of antibiotic pollution, and (6) customers of corns, corn products, and corn-fed animal products. NJIT's undergraduate body is very diverse, and its 8008 student body is 65% nonwhite. This project will allow PI to host students from disadvantaged backgrounds through different programs at NJIT (e.g., Summer Intern Program and Undergraduate Research Initiatives) to perform laboratory research. To date, the PI's lab has hosted eleven undergraduate students (ten are female or non-white) and seven high school students (six are female or non-white). The PI is also motivated to disseminate our novel research findings integrating the streamlined scientific principles and advanced remediation innovations at local K-12 programs and science café (meetup events for junior science lovers) or via podcast targeting teenagers and other targeted audiences. This project sponsors a postdoc researcher to primarily work on the laboratory work. Accordingly, we have recruited Dr. Qiong Wu, a female young scientist with outstanding expertise in agricultural microbiology. Changes/Problems:Due to the pandemic, our lab was closed between March and July 2020, which has delayed the experimental research. We thus shifted the focus to metagenomic analyses that rely on computation efforts. What opportunities for training and professional development has the project provided?This project engages the training and participation of one postdoc, Dr. Qiong Wu, and one PhD student, Dung Ngoc Pham. How have the results been disseminated to communities of interest?We published a Research Article on the acclimation of SA resistance genes and associated bacteria on microplastics in the Journal of Hazardous Materials Letters (DOI: 10.1016/j.hazl.2021.100014). Due to the pandemic, no presentations were given at domestic conferences as planned. What do you plan to do during the next reporting period to accomplish the goals?(1) We plan to sequence the genome and metagenomes of the SA-degrading cultures we obtained and further uncover the molecular foundations for SA inactivation. (2) We will focus on using bioinformatics to unravel the diversity and distribution of ARGs in the maize rhizosphere on a global scale.

Impacts
What was accomplished under these goals? (1) We collected a number of environmental samples, including agricultural soils and activated sludge. Using the conventional dilution and plating method, we obtained a gram-negative bacterium, Xanthobacter sp. LD2, and a consortium RD1, which both carry the sulfonamide monooxygenase gene sadA through the enrichment of activated sludge samples. SA inactivation activities of these cultures were validated in batch assays. Metabolites of SA degradation were screened and identified using HPLC. These cultures also contain the sul1 gene that confers resistance to SAs at high concentrations. (2) We identified 52 metagenomes of maize rhizosphere available in the databases of Joint Genome Institute (JGI, https://genome.jgi.doe.gov/portal/) and National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra). All metagenomes were obtained by the Illumina sequencing-by-synthesis technology, resulting in the assembly-based dataset sizes of 0.4~7.6 Gb. Among them, 27 metagenomes for maize rhizosphere samples collected in Michigan (15) and California (12) were assembled and downloaded directly from JGI. For the other 25 metagenomes in South Africa (21) and the Netherland (4), only raw sequencing reads were available from NCBI SRA. Trimmed metagenomic reads were then assembled into contigs using SPAdes v3.13 with the metaSPAdes option and default parameter settings. (3) We conducted a microcosm assay to investigate the abundance and dynamics of SA resistance genes in the plastisphere, which refers to the biofilms attached to microplastics. We demonstrated both polyethylene (PE) and polystyrene (PS) microplastics can acclimate biofilms enriched with SA resistance genes (sul1 and sul2) in comparison with fine sands as control particles. Particularly, the abundance of sul1 was positively associated with the mobile genetic element (intI1), implying the mobility of this ARG. Absolute abundances of these genes were further elevated by 1.2∼4.5 fold when sulfamethoxazole was initially spiked as a representative sulfonamide. The combination of 16S rRNA amplicon sequencing and differential ranking analysis revealed that microplastics selectively promoted antibiotic-resistant and pathogenic taxa (e.g., Raoultella ornithinolytica and Stenotrophomonas maltophilia) with enrichment indices ranging from 1.6 to 3.3. Furthermore, heterotrophic Novosphingobium and filamentous Flectobacillus accounted for 14.6 % and 3.3 % on average in microplastic biofilms, respectively, which were up to 2.8 and 11.1 times higher than those in sand biofilms. Dominance of these bacterial species may contribute to initial biofilm formation that facilitates subsequent colonization and proliferation of ARB and pathogens, thus amplifying their risks in the receiving environments and beyond.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Pham, Dung Ngoc, Lerone Clark, and Mengyan Li. "Microplastics as hubs enriching antibiotic-resistant bacteria and pathogens in municipal activated sludge." Journal of Hazardous Materials Letters 2 (2021): 100014.