The Effects of Microplastics on antibiotic resistance genes in

landfill Ecosystem

Name of the PhD Candidate

Rakia Manzoor
Young Researcher,
rmanzoor451@gmail.com
Tel: +923009660106

Name and Designation of Supervisor

Prof. Dr. Liyan Song
School of Resource and Environmental Engineering
Anhui University
Email: songliyan@cigit.ac.cn
Introduction
Recent studies have shown that an increased amount of plastic waste is released into the
environment as a result of the constant growth in plastic manufacturing and usage. These
plastic materials, which include macroplastics (with a diameter of >5 mm), microplastics
(MPs, 5 mm), and nanoplastics. MPs have been widely found in a variety of ecosystems
including rivers, soils, wastewater treatment facilities and landfills (Shi et al, 2020). Even
though research on the effects of microplastic pollution is increasing, still little is known
about their direct effects, which appear to be a threat to human health given the spread
of disease and antibiotic resistance genes (Sun et al, 2021).
Microplastic pollution is alarming global problem. These particles offer a hydrophobic
surface that serves as a substrate for microbial attachment and easily promotes the
development of microbial biofilms. These biofilms contain pathogenic bacteria, including
opportunistic pathogens. Additionally, it has been found that some of these pathogens
are multidrug resistant. Microplastics are known to facilitate horizontal gene transfer in
bacteria, which may aid in the spread of antibiotic resistance (Kabir et al, 2023)..
Landfill ecosystem is an important reservoir for studying the effect of microplastics on
microbial drug resistance (ARGs). Landfill ecosystem contain a variety of pollutants,
including complex microbial consortiums, persistent organic compounds, and anaerobic
degradation processes, which promote the occurrence, growth, and transmission of
ARGs and antibiotic-resistant bacteria (ARB). Recent studies have shown that antibiotic
resistance genes could escape into the local ecosystem through landfills and leachate
posing serious health threat to all living organisms. Although there is an ongoing research
on the origins and distribution of antibiotics and ARGs in landfill ecosystem, the direct
effects of microplastics, particularly in relation to the ARGs have not been thoroughly
studied yet. This study will discuss the impact of MPs on ARGs and provide an overview
of established studies on antibiotics and ARGs in landfills (Silva et al, 2021)..
Graphical Abstract
Cellular level

5
MPs 4
3
2
1
0

Increased
increased
bacterial Increased
ROS ARGs
abundance cell
Leachate abundance
membrance
permeability

Importance of the study

Microplastics have the potential to upset the balance of organism populations, and any
negative effects on a single species may have unintended ramifications for the ecosystem
as a whole. Ecosystems and their health can thus be plainly damaged by microplastics
through a number of processes. ARGs are becoming a major threat to human health on
a global scale. The precise impact of MPs on the incidence of ARGs, however, is not
understood. An earlier study showed that MPs had an impact on bacterial community
evolution, which is essential to the variances in ARG profiles. According to Sun et al., the
production of reactive oxygen species by NPs can prevent the growth of bacteria (ROS)
(Wan et al, 2022; Shi et al, 2020).The horizontal transmission of ARGs may be facilitated
by the ROS because they can make cell membranes more permeable. For a better
understanding of the combined impacts of MPs and ARGs, it is crucial to investigate these
effects and the underlying mechanisms.
Objective of this study
In this study we will use polystyrene particles of three various sizes will introduce these
particles to raw landfill leachates to examine how they affect ARGs. In both short- and
long-term MP exposure trials, the bacterial population and cell membrane permeability
will be examined.
The goals of this study are to:
(I) track the development of leachate-ARGs following exposure to N/MPs; and
(2) Investigate the underlying mechanisms from the perspective of bacterial diversity at
the community and cellular levels.
Sample collection
Samples of leachate will be taken from the Anhui landfill near Anhui, China. Every day,
roughly 12k tonnes of waste, largely plastic garbage, are brought to the landfill.
In order to preserve the sampled leachates, glass serum vials will be cleaned and covered
with aluminium foil before use. Within 12 hours of sample collection, all of the serum vials
will be collected in laboratory and stored in ice boxes. Anhui Biochemical Technology Co.
Ltd. Will provide the polystyrene plastic needed for this experiment in the form of a white
bead suspension (2.5% w/v) (Anhui, China). The polymeric Conformation of MPs will be
verified by infrared spectroscopy.
Exposure experiments
Leachate filtrate samples will be thoroughly combined in a sizable sterilized glass
container before being evenly distributed into each testing bottle of sterilized serum,
ensuring that the leachate will initially be in the same condition for each treatment group.
At a subsequent exposure dosage of 400 mg L1, the purchased polystyrene beads will
be added. Without using any polystyrene beads, the control samples were given the same
treatment.
Biochemical and sequence analysis
Using a protocol for DNA extraction, the whole DNA will be extracted in accordance with
the manufacturer's instructions. A UV-vis spectrophotometer will be used to assess the
yields and quality of the isolated DNA. A real-time quantitative polymerase chain reaction
(qPCR) assay will be used to find out various ARGs groups. We proposed the presence
of eight different ARG types, including sulfonamide, aminoglycoside and beta-lactam
resistance gene. In addition to this, 16S rRna sequencing will be performed. a cell-
permeant ROS indicator, will also be used to measure intracellular ROS levels utilizing a
fluorescence-based technique.
Statistical analysis
Expected results following outcomes are expected to be acquired at the end of this
research: We will be able to functionally confirm the association between ARGs and MPs
that is not well-established so far. Also we will able to access the expression analysis of
AGRs with the progression of MP concentration by developing different assay
Expected results
Following outcomes are expected to be acquired at the end of this research:
1. We will be able to functionally confirm the association between ARGs and MPs
2. We will be able to validate the Effects of MPs on bacterial communities.
3. Also we will able to analysis the Effects of MPs on the intracellular ROS production
and Cell membrane permeability
References
 Shi, J., Wu, D., Su, Y., & Xie, B. (2020). (Nano) microplastics promote the
propagation of antibiotic resistance genes in landfill leachate. Environmental
Science: Nano, 7(11), 3536-3546.
 Sun, J., Zhu, Z. R., Li, W. H., Yan, X., Wang, L. K., Zhang, L., ... & Ni, B. J. (2021).
Revisiting microplastics in landfill leachate: unnoticed tiny microplastics and their
fate in treatment works. Water Research, 190, 116784.
 Kabir, M. S., Wang, H., Luster-Teasley, S., Zhang, L., & Zhao, R. (2023).
Microplastics in landfill leachate: Sources, detection, occurrence, and
removal. Environmental Science and Ecotechnology, 100256.
 Silva, A. L., Prata, J. C., Duarte, A. C., Soares, A. M., Barceló, D., & Rocha-Santos,
T. (2021). Microplastics in landfill leachates: The need for reconnaissance studies
and remediation technologies. Case Studies in Chemical and Environmental
Engineering, 3, 100072.
 Wan, Y., Chen, X., Liu, Q., Hu, H., Wu, C., & Xue, Q. (2022). Informal landfill
contributes to the pollution of microplastics in the surrounding
environment. Environmental Pollution, 293, 118586.

Previous work of the applicant

2017-2020 (IGDB CAS as a Researcher) Research Summary
N6-methyladenosine (m6A) is the most abundant messenger RNA modification present
in eukaryotes and deposited by m6A writers, erasers and readers. The writer complex is
known to contain the methyl transferases METTL3 and METTL14 and WTAP. Despite
the recent studies revealing the role of m6A in regulating cognitive behavior of animals,
epitranscriptomic regulation of social behavior by m6A modification remains unclear. Here
we found that dysregulation of m6A in the neocortex of mouse brain impairs social
behavior of animals. Further analyses show that excitatory-inhibitory (E-I) balance, spine
formation and synaptic protein expression in prefrontal cortex are disrupted by loss of
Mettl14. Unexpectedly, RNAseq and m6Aseq reveal the dysregulation of potassium
channels (Kcnq1, Kcnq3 and Kcnq5) in both prefrontal cortex and hippocampus of m6A-
deficient mice. This study suggests E-I imbalance by deficient m6A modification as a
causative mechanism implicated in potassium channelopathy and autism spectrum
disorders (ASDs).
FIGURE 2: Epitranscriptomic Regulation of m6A deficient mice leads to autistic features.

2015-2017 (NIBGE as a Research Associate)

Research Summary
β thalassemia is the most common hereditary monogenic disorder in the world with an
overall carrier rate of 1.5%. In Pakistan, it is the most prevalent genetically transmitted
blood disease with a carrier rate of 5–7%; approximately 4000 new births are identified
annually. A total of more than 100 families were screened for five most prevalent
mutations causing β thalassemia in Pakistani population. Allele- specific polymerase
chain reaction (ASPCR) was performed for the characterization of these mutations. After
the confirmation of mutation, Chorionic Villus Samples (CVS) taken from carrier mother
were screened for mutations. The results of the study provided information about mutation
spectrum of β thalassemia in local population. Carrier screening, genetic counseling and
prenatal diagnosis are the most effective approaches to prevent further affected births.
Genetic testing, coupled with prenatal diagnosis, genetic counseling and mass
awareness will greatly reduce the incidence of β thalassemia in Pakistan.
2013-2015 (M.phil)
Research Summary
Although genetic disorders are rare, they appear with severe conditions. Genetic
disorders which are transmitted through generations in autosomal recessive fashion are
caused by the inheritance of a mutant allele from each parent by the child. Congenital
non syndromic hearing impairment is an example of such disorders characterized by
compromised ability to hear. It is a highly heterogeneous disorder wherein 80% of the
disease genes are involved in autosomal recessively inherited non- syndromic hearing
impairment. In this study, four families, A-D, had patients affected with congenital

non syndromic hearing impairment, these families were ascertained from different regions
of Pakistan. Information about families and mode of inheritance was established by
interviewing family history from the elder members. Genomic DNA was extracted from
the venous blood collected from affected persons, their normal siblings and parents.
Highly polymorphic STR (Short Tandem Repeats) markers were used to map the
candidate causative loci/genes by homozygosity mapping. Results of families A- D
showed no disease specific homozygous region around the candidate causative loci, all
the families were excluded to these loci/genes. Exclusion of known loci demonstrate that
there are yet unidentified causative loci or genes to be explored. From these results, we
may also infer the possibility of compound heterozygosity playing a role; a condition
wherein families may have two different mutations on the two homologous chromosomes.

List of Publications as co-author

1. Hamid, N., Junaid, M., Manzoor, R., Jia, P.P., and Pei, D.S. (2020). Prioritizing
phthalate esters (PAEs) using experimental in vitro/vivo toxicity assays and computational
in silico approaches. J Hazard Mater 398, 122851. (IF=8.00)
2. Mehmood, S., Bilal, M., Manzoor, R., and Iqbal, H.M.N. (2019). Deciphering the
adult brain development complexity by single-cell transcriptome analysis—a review.
Materials Today Chemistry 13, 88-97. (IF=3.70)
3. Nazir, A., Zhao, Y., Li, M., Manzoor, R., Tahir, R.A., Zhang, X., Qing, H., and Tong,
Y. (2020).
Structural Genomics of repA, repB 1-Carrying IncFIB Family pA1705-qnrS, P911021-
tetA, and P1642-tetA, Multidrug-Resistant Plasmids from Klebsiella pneumoniae. Infect
Drug Resist 13, 1889-1903. (IF=2.98)