Syed A Hashsham

• PhD
• Professor (Full) at Michigan State University

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-...

Electrochemical approaches, along with miniaturization of electrodes, are increasingly being employed to detect and quantify nucleic acid biomarkers. Miniaturization of the electrodes is achieved through the use of screen-printed electrodes (SPEs), which consist of one to a few dozen sets of electrodes, or by utilizing printed circuit boards. Elect...

Environmental pollution is escalating due to rapid global development that often prioritizes human needs over planetary health. Despite global efforts to mitigate legacy pollutants, the continuous introduction of new substances remains a major threat to both human and ecosystem health. In response, global initiatives are focusing on risk assessment...

The presence and spread of antimicrobial resistance genes (ARGs) in soil represent a significant threat to human health. However, the ARGs harbored by antibiotic-degrading microorganisms in soil are inadequately characterized. Employing diverse molecular techniques, we investigate the dynamics of ARGs, mobile genetic elements, and virulence factors...

The emergence and rapid spread of antimicrobial resistance is of global public health concern. The gut microbiota harboring diverse commensal and opportunistic bacteria that can acquire resistance via horizontal and vertical gene transfers is considered an important reservoir and sink of antibiotic resistance genes (ARGs). In this review, we descri...

Antibiotic resistance genes (ARGs) and human pathogenic bacteria (HPB) in leafy vegetable is a matter of concern as they can be transferred from soil, atmosphere, and foliar sprays, and poses a potential risk to public health. While traditional disinfection technologies are effective in reducing the presence of ARGs and HPB in soil. A new technolog...

Wildlife monitoring programs are instrumental for the assessment of species, habitat status, and for the management of factors affecting them. This is particularly important for species found in freshwater ecosystems, such as amphibians, as they have higher estimated extinction rates than terrestrial species. We developed and validated two species-...

The effect of manure application on the distribution and accumulation of antibiotic resistance genes (ARGs) in tissue of root vegetables remains unclear, which poses a bottleneck in assessing the health risks from root vegetables due to application of manure. Towards this goal, experiments were conducted in pots to investigate the distribution and...

Segmented filamentous bacteria (SFB) and Bacteroides fragilis are known to interact with the host immune response through the aryl hydrocarbon receptor (Ahr). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxicant and a high-affinity Ahr ligand has the potential to modify the effect of SFB and B. fragilis. MicroRNAs (miRNA) with thei...

Urban wastewater systems (UWSs) are a main receptacle of excreted antibiotic resistance genes (ARGs) and their host microorganisms. However, we lack integrated and quantitative observations of the occurrence of ARGs in the UWS to characterize the sources and identify processes that contribute to their fate. We sampled the UWSs from three medium-siz...

In this work, we made an effort by differing the concentration of indium (In³⁺) ions doped in copper bismuthate (CuBi2O4) nanoparticles (NPs) through one step hydrothermal process for selective and sensitive electrochemical detection of antipsychotic drug chlorpromazine (CPZ). Results highlight that In³⁺ doped CuBi2O4 is more suitable to promotes t...

Integrated and quantitative observations of antibiotic resistance genes (ARGs) in urban water systems (UWSs) are lacking. We sampled three UWSs for clinically important extended spectrum β-lactamase (ESBL) and carbapenemase (CP) genes, mobile genetic elements and microbial communities. Sewage – especially from hospitals – carried substantial loads...

The persistence of antibiotic resistance genes (ARGs) under the aerobic vs. anaerobic conditions is unknown, especially under different fertilization. Towards this goal, a microcosm experiment was carried out with chemical fertilized and manured soil under aerobic and anaerobic conditions. High throughput qPCR was used to analyze ARGs with 144 prim...

Microbial contamination in water remains a global issue in threatening human health. Although current disinfection methods can effectively control microbial contamination, the formation of harmful disin-fection byproducts is increasingly questioned. Towards this goal, a facile, green and low-priced bacte-riocide-magnetic biochar/quaternary phosphon...

Microbial contamination in water remains a global issue in threatening human health. Although current disinfection methods can effectively control microbial contamination, the formation of harmful disinfection byproducts is increasingly questioned. Towards this goal, a facile, green and low-priced bacteriocide - magnetic biochar/quaternary phosphon...

We examined the bacterial endophyte–enriched root-associated microbiome within rice (Oryza sativa) 55 days after growth in soil with and without urea fertilizer and/or biofertilization with a growth-promotive bacterial strain (Rhizobium leguminosarum bv. trifolii E11). After treatment to deplete rhizosphere/rhizoplane communities, washed roots were...

Different long-term fertilization regimes may change indigenous microorganism diversity in the arable soil and thus might influence the persistence and transmission of manure-born antibiotic resistance genes (ARGs). Different manure origins and composting techniques might affect the fate of introduced ARGs in farmland. A four-month microcosm experi...

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-...

Shotgun sequencing was used for the quantification of taxonomic and functional biomarkers associated with chlorinated solvent bioremediation in twenty groundwater samples (five sites), following bioaugmentation with SDC-9. The analysis determined the abundance of 1) genera associated with chlorinated solvent degradation, 2) reductive dehalogenase (...

Different fertilization and cropping systems may influence short- and long-term residues of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) in soil. Soils from dryland (peanut) and paddy (rice) fields, which originated from the same non-agricultural land (forested), were treated with either chemical fertilizer, composted manur...

This review su mmarizes selected publications from 2017 highlighting the occurrence of antimicrobial resistance (AMR) genes in the environment with emphasis on the aquatic environment. The review also covers different treatment technologies being developed for AMR genes as an environmental contaminant. The progress made in the area of AMR gene data...

The high-throughput antibiotic resistance gene (ARG) qPCR array, initially published in 2012, is increasingly used to quantify resistance and mobile determinants in environmental matrices. Continued utility of the array; however, necessitates improvements such as removing or redesigning questionable primer sets, updating targeted genes and coverage...

Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending o...

Organ-on-chip systems are the emerging in vitro tools to study the effect of chemicals, pharmaceuticals, and nanomaterials on various organs. They combine key functional characteristics of various human organs with specialized microfluidic platforms to evolve systems that mimic the biochemical, metabolic, genetic, and functional characteristics of...

Following decades of ecologic and economic impacts from a growing list of nonindigenous and invasive species, government and management entities are committing to systematic early- detection monitoring (EDM). This has reinvigorated investment in the science underpinning such monitoring, as well as the need to convey that science in practical terms...

Loop-mediated isothermal amplification (LAMP) of aquatic invasive species environmental DNA (AIS eDNA) was used for rapid, sensitive, and specific detection of Dreissena sp. relevant to the Great Lakes (USA) basin. The method was validated for two uses including i) direct amplification of eDNA using a hand filtration system and ii) confirmation of...

Results from the experimental specificity analysis. (TIF)

Results from the theoretical specificity analysis as conducted using BLAST. Individual primers were evaluated for specificity by analyzing max score, % query coverage, E value, and % identity. (XLSX)

Direct amplification results. An excel datasheet is included with the results from direct amplification of all environmental samples. (XLSX)

Activated carbon (AC) is an increasingly attractive remediation alternative for the sequestration of dioxins at contaminated sites globally. However, the potential for AC to reduce the bioavailability of dioxins in mammals and the residing gut microbiota has received less attention. This question was partially answered in a recent study examining 2...

This review summarizes selected publications of 2016 with emphasis on occurrence and treatment of antibiotic resistance genes and bacteria in the aquatic environment and wastewater and drinking water treatment plants. The review is conducted with emphasis on fate, modeling, risk assessment and data analysis methodologies for characterizing abundanc...

The remediation of chlorinated solvent contaminated sites frequently involves bioaugmentation with mixed cultures containing Dehalococcoides mccartyi. Their activity is then examined by quantifying reductive dehalogenase (RDase) genes. Recently, we described a rapid, low cost approach, based on loop mediated isothermal amplification (LAMP), which a...

The gut microbiome is an important modulator of host gene expression, impacting important functions such as the innate immune response. Recent evidence suggests that the inter-domain communication between the gut microbiome and host may in part occur via microRNAs (small, non-coding RNA molecules) which are often differentially expressed in the pre...

Environmental toxicants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR), are known to induce host toxicity and structural shifts in the gut microbiota. Key bacterial populations with similar or opposing functional responses to AhR ligand exposure may potentially help regulate expression of genes associated wit...

Antimicrobial resistance genes (ARGs) present in the environment pose a risk to human health due to potential for transfer to human pathogens. Surveillance is an integral part of mitigating environmental dissemination. Quantification of the mobile genetic element class 1 integron-integrase gene (intI1) has been proposed as a surrogate to measuring...

TaqMan probe-based quantitative polymerase chain reaction (qPCR) specific to the biomarker reductive dehalogenase (RDase) genes is a widely accepted molecular biological tool (MBT) for determining the abundance of Dehalococcoides sp. in groundwater samples from chlorinated solvent-contaminated sites. However, there are significant costs associated...

Microfluidic DNA biochips capable of detecting specific DNA sequences are useful in medical diagnostics, drug discovery, food safety monitoring and agriculture. They are used as miniaturized platforms for analysis of nucleic acids‐based biomarkers. Binding kinetics between immobilized single stranded DNA on the surface and its complementary strand...

MicroRNAs have been proposed to be a class of biomarkers of disease as expression levels are significantly altered in various tissues and body fluids when compared to healthy controls. As such, the detection and quantification of microRNAs is imperative. While many methods have been established for quantification of microRNAs, they typically rely o...

Guidelines and regulations to control Legionella pneumophila in cooling water systems of large buildings are evolving due to the increasing number of outbreaks. Rapid, on-site, simple, and sensitive quantification methods that are also able to assess viability may be extremely useful in monitoring and control. Culture-based methods for measuring L....

Dysbiosis of the gut microbiome via antibiotics, changes in diet, and infection can select for bacterial groups that more frequently harbor antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). However, the impact of environmental toxicants on the reservoir of ARGs in the gut microbiome has received less attention. 2,3,7,8-tetra...

Parallel detection approaches are of interest to many researchers interested in identifying multiple water and foodborne pathogens simultaneously. Availability and cost-effectiveness are two key factors determining the usefulness of such approaches for laboratories with limited resources. In this study, we developed and validated a high-density mic...

Virulence factor activity relationships (VFARs) - a concept loosely based on quantitative structure-activity relationships (QSARs) for chemicals was proposed as a predictive tool for ranking risks due to microorganisms relevant to water safety. A rapid increase in sequencing capabilities and bioinformatics tools has significantly increased the pote...

Antimicrobial resistance (AMR) is recognized as a global threat to human health. Rapid detection and characterization of AMR is a critical component of most antibiotic stewardship programs. Methods based on amplification of nucleic acids for detection of AMR are generally faster than culture-based approaches but they still require several hours to...

We are reporting a most probable number approach integrated to loop mediated isothermal technique (MPN-LAMP) focusing on Gram-negative Escherichia coli and Gram-positive Enterococcus faecalis bacterial cells without nucleic acids extraction. LAMP assays for uidA from E. coli and gelE from E. faecalis were successfully performed directly on cells up...

Soil is an important environmental reservoir of antibiotic resistance genes (ARGs), which are increasingly recognized as environmental contaminants. Methods to assess the risks associated with the acquisition or transfer of resistance mechanisms are still underdeveloped. Quantification of background levels of antibiotic resistance genes and what al...

Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groun...

This review summarizes important publications from 2015 pertaining to the occurrence of antimicrobial resistance (AMR) in the environment. Emphasis is placed on sources of antibiotic resistance in the aquatic environment including wastewater treatment plants, hospitals, and agriculture, treatment and mitigation techniques, and surveillance and anal...

Sphingomonas wittichii strain RW1 (RW1) is one of the few strains that can grow on dibenzo-p-dioxin (DD). We conducted a transcriptomic study of RW1 using RNA-Seq to outline transcriptional responses to DD, dibenzofuran (DF), and the smectite clay mineral saponite with succinate as carbon source. The ability to grow on DD is rare compared to growth...

Genes differentially expressed between DD, and the early slow growth phase culture DD1. Colors as in S1 Table. (DOCX)

Expression changes of genes involved in cell motility under SAP, DD, and DF relative to SUC. Down-regulated genes are marked in red background, genes without significant change are marked in tan background. (COG N: Cell motility, COG M: Cell wall/membrane/envelope biogenesis; COG O: Post-translational modification, protein turnover, chaperones; COG...

Growth curves of RW1 cell cultures used for transcriptomes. RW1 cell cultures growing on (A) succinate as sole carbon source; (B) succinate as sole carbon source and in the presence of 0.7% Cs-Saponite; (C) 3 mM dibenzo-p-dioxin as sole carbon; (D) 3 mM dibenzofuran as sole carbon source. (Note one replicate of DD had delayed, biphasic growth). (TI...

Upper pathway of dibenzofuran/dibenzo-p-dioxin degradation. dbfB was up-regulated (*†) in response to both dibenzofuran (DF) and dibenzo-p-dioxin (DD). (TIFF)

Differentially expressed genes between SUC, DD, and DF. Statistically significant differences are in a green background for increased, red for decreased; differences that are not statistically significant are in grey background with a green font for an increase and a red font for a decrease. (DOCX)

Scanning Electron Microscopy (SEM) images of RW1 cells. (A) SUC—cells self-aggregate, produce expolysaccharide and slime; (B) SAP—cells appear free-floating and did not aggregate or produce slime, but were attracted to smaller clay particles; (C) DD and (D) DF—cells form large aggregates, attached to DF crystals. White bar is 100 μm. Inset shows la...

IMPORTANCE Agricultural antibiotic use results in clusters of cooccurring resistance genes that together confer resistance to multiple antibiotics. The use of a single antibiotic could select for an entire suite of resistance genes if they are genetically linked. No links to bacterial membership were observed for these clusters of resistance genes....

Performance of the qPCR primer set and the 16S rRNA V4 degenerate primer set in amplifying Bacteroidetes. When the temperature was high, the V4 primer did not amplify Bacteroidetes efficiently. Download

Sequence diversity of all gene types across all the samples. There are 40 rows next to each gene name indicating the 40 most abundant alleles, sorted by abundance, shown as percent abundance in each sample. The shading to indicate the allele abundance is based on a log scale. Sample triplicates are separated by vertical gray lines. Horizontal black...

Primer sets used in this study. Primer name indicates the targeted gene.

Heat map of the averaged qPCR results. Both axes are clustered based on Euclidean distance. Samples cluster largely by sample type. The feral samples are the most distinct of all samples. intI1 was largely not detected in the NADC or feral animals. Download

Examples of genes from the intI1-IS6100 resistance cluster in known genomes. Genes with bold black outlines are identical to alleles of the intI1-IS6100 cluster. The same integron and cassettes were found in two E. coli strains, including IS6100, as the terminal cassette. A similar intI1-IS6100 arrangement was found in the Klebsiella strain. Downlo...

qPCR threshold cycle results for all primer sets and samples used in our analysis.

Results are shown including validation of the 4-primer amplification chemistry with our primers, validation of the qPCR results by comparison with previous results on the Open Array platform (Applied Biosystems), amplicon specificity and diversity, and detection of Bacteroidetes by the V4 16S rRNA primer set. Download

Allele-specific relative abundance compared to 16S rRNA abundance. Each gene listed represents the most abundant allele of that gene from each of the different farms, Beijing (A), Jiaxing (B), and Putian (C), and the NADC and feral animals (D). Colored highlights indicate genes belonging to the intI1-IS6100 cluster (blue), the IS1216-tet cluster (g...

Success rate of assembly of the 47 amplicon sets. The percentage of the raw forward and reverse reads that were successfully assembled was nearly 100% when the entire amplicon was sequenced by both the forward and reverse reads. When the overlap between the forward and reverse reads is shorter, the percentage of the reads that can be assembled drop...

Percent identity of the assembled amplicons to the reference sequences. Assembled amplicons were mapped to reference sequences as part of the FrameBot software to correct frameshift errors. From this analysis, we observed high percent identity of the obtained amplicon reads to the reference sequences. Each of the thin lines represents the percentag...

Real time quantitative polymerase chain reaction (qPCR) protocols specific to the reductive dehalogenase (RDase) genes vcrA, bvcA, and tceA are commonly used to quantify Dehalococcoides spp. in groundwater from chlorinated solvent contaminated sites. In this study, loop mediated isothermal amplification (LAMP) was developed as an alternative approa...

An antibiotic resistance (AR) Dashboard application is being developed regarding the occurrence of antibiotic resistance genes (ARG) and bacteria (ARB) in environmental and clinical settings. The application gathers and geospatially maps AR studies, reported occurrence, and antibiograms, which can be downloaded for offline analysis. With the integr...

Loop-mediated isothermal amplification (LAMP) is increasingly used for point-of-care nucleic acid based diagnostics. LAMP can be monitored in real-time by measuring the increase in fluorescence of DNA binding dyes. However, there is little information comparing the effect of various fluorescent dyes on signal to noise ratio (SNR) or threshold time...

A microfluidic card is described for simultaneous and rapid genetic detection of multiple microbial pathogens. The hydrophobic surface of native acrylic and a novel microfluidic mechanism termed “airlock” were used to dispense sample into a series of 64 reaction wells without the use of valves, external pumping peripherals, multiple layers, or vacu...

In a clinical setting, molecular assays such as polymerase chain reaction offer a rapid means to infer or confirm identity and therapeutic decisions. Accordingly, a number of molecular assays targeting identity and antibiotic resistance (AR) genes have been developed; however, these methods can be technically complex and relatively expensive. Herei...

##Assembly-Data-START## Sequencing Technology :: Sanger dideoxy sequencing ##Assembly-Data-END##

Crossflow membrane filtration is emerging as a concentration method for the recovery and detection of viruses in large volumes of water. To increase virus recovery, an ultrafiltration membrane is usually "blocked" with a proteinaceous solution or a chemical dispersant. This proof-of-concept study explores a novel approach using controlled and rapid...

Nucleic acid amplification of biomarkers is increasingly used to measure microbial activity and predict remedial performance in sites with Trichloroethene (TCE) contamination. Field-based genetic quantification of microorganisms associated with bioremediation may help increase accuracy that is diminished through transport and processing of groundwa...

This review summarizes some key literature published in 2013 that pertains to the detection and occurrence of pathogens and indicators of waterborne pathogens. Particular emphasis was placed on emerging detection methods that when fully developed may have an impact on the application of molecular tools to characterize the microbial quality of water...

Decentralized, bedside, or point of care analysis of nucleic acids (DNA/RNA/mRNA/microRNA)-based markers is expected to be a key component of digital healthcare. Nucleic acids-based approaches allow for screening of disease and pathogens, disease surveillance, selection of treatment, treatment effectiveness, differential diagnosis, risk assessment,...

Sewage pollution remains the most significant source of human waterborne pathogens. This study describes the detection and characterization of human enteric viruses in community wastewaters using cell culture coupled with multiple target microarrays (with a total of 780 unique probes targeting 27 different groups of both DNA and RNA viruses) and po...

This review summarizes the literature published in 2012 pertaining to the occurrence and detection of fecal indicator bacteria (FIB) and pathogens. It is organized into the following sections: i) methods for the detection and quantification of waterborne fecal indicators and pathogens including polymerase chain reaction (PCR), isothermal amplificat...

Antibiotic resistance genes (ARGs) are emerging contaminants posing a potential worldwide human health risk. Intensive animal husbandry is believed to be a major contributor to the increased environmental burden of ARGs. Despite the volume of antibiotics used in China, little information is available regarding the corresponding ARGs associated with...

Fomites are known to play a role in the transmission of pathogens. Quantitative analysis of the parameters that affect sample recovery efficiency (SRE) at the limit of detection of viruses on fomites will aid in improving quantitative microbial risk assessment (QMRA) and infection control. The variability in SRE as a function of fomite type, fomite...