Genomics of Environmental Salmonella: Engaging Students in the Microbiology and Bioinformatics of Foodborne Pathogens

We have developed and implemented an undergraduate microbiology course in which students isolate, characterize, and perform whole genome assembly and analysis of Salmonella enterica from stream sediments and poultry litter. In the development of the course and over three semesters, successive teams of undergraduate students collected field samples and performed enrichment and isolation techniques specific for the detection of S. enterica. Eighty-eight strains were confirmed using standard microbiological methods and PCR of the invA gene. The isolates’ genomes were Illumina-sequenced by the Center for Food Safety and Applied Nutrition at the FDA and the Virginia state Division of Consolidated Laboratory Services as part of the GenomeTrakr program. Students used GalaxyTrakr and other web- and non-web-based platforms and tools to perform quality control on raw and assembled sequence data, assemble, and annotate genomes, identify antimicrobial resistance and virulence genes, putative plasmids, and other mobile genetic elements. Strains with putative plasmid-borne antimicrobial resistance genes were further sequenced by students in our research lab using the Oxford Nanopore MinIONTM platform. Strains of Salmonella that were isolated include human infectious serotypes such as Typhimurium and Infantis. Over 31 of the isolates possessed antibiotic resistance genes, some of which were located on large, multidrug resistance plasmids. Plasmid pHJ-38, identified in a Typhimurium isolate, is an apparently self-transmissible 183 kb IncA/C2 plasmid that possesses multiple antimicrobial resistance and heavy-metal resistance genes. Plasmid pFHS-02, identified in an Infantis isolate, is an apparently self-transmissible 303 kb IncF1B plasmid that also possesses numerous heavy-metal and antimicrobial resistance genes. Using direct and indirect measures to assess student outcomes, results indicate that course participation contributed to cognitive gains in relevant content knowledge and research skills such as field sampling, molecular techniques, and computational analysis. Furthermore, participants self-reported a deeper interest in scientific research and careers as well as psychosocial outcomes (e.g., sense of belonging and self-efficacy) commonly associated with student success and persistence in STEM. Overall, this course provided a powerful combination of field, wet lab, and computational biology experiences for students, while also providing data potentially useful in pathogen surveillance, epidemiological tracking, and for the further study of environmental reservoirs of S. enterica.

Download Full-text

Detection of mobile genetic elements associated with antibiotic resistance in Salmonella enterica using a newly developed web tool: MobileElementFinder

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa390 ◽

2020 ◽

Vol 76 (1) ◽

pp. 101-109 ◽

Cited By ~ 1

Author(s):

Markus H K Johansson ◽

Valeria Bortolaia ◽

Supathep Tansirichaiya ◽

Frank M Aarestrup ◽

Adam P Roberts ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Salmonella Enterica ◽

Sequence Data ◽

Sequence Similarity ◽

Mobile Genetic Elements ◽

Metal Resistance ◽

Animal Origin ◽

Genetic Elements ◽

Antimicrobial Resistance Genes

Abstract Objectives Antimicrobial resistance (AMR) in clinically relevant bacteria is a growing threat to public health globally. In these bacteria, antimicrobial resistance genes are often associated with mobile genetic elements (MGEs), which promote their mobility, enabling them to rapidly spread throughout a bacterial community. Methods The tool MobileElementFinder was developed to enable rapid detection of MGEs and their genetic context in assembled sequence data. MGEs are detected based on sequence similarity to a database of 4452 known elements augmented with annotation of resistance genes, virulence factors and detection of plasmids. Results MobileElementFinder was applied to analyse the mobilome of 1725 sequenced Salmonella enterica isolates of animal origin from Denmark, Germany and the USA. We found that the MGEs were seemingly conserved according to multilocus ST and not restricted to either the host or the country of origin. Moreover, we identified putative translocatable units for specific aminoglycoside, sulphonamide and tetracycline genes. Several putative composite transposons were predicted that could mobilize, among others, AMR, metal resistance and phosphodiesterase genes associated with macrophage survivability. This is, to our knowledge, the first time the phosphodiesterase-like pdeL has been found to be potentially mobilized into S. enterica. Conclusions MobileElementFinder is a powerful tool to study the epidemiology of MGEs in a large number of genome sequences and to determine the potential for genomic plasticity of bacteria. This web service provides a convenient method of detecting MGEs in assembled sequence data. MobileElementFinder can be accessed at https://cge.cbs.dtu.dk/services/MobileElementFinder/.

Download Full-text

322. Evaluation of the BioFire® Bone and Joint Infection (BJI) Panel for the Detection of Microorganisms and Antimicrobial Resistance Genes in Synovial Fluid Specimens

Open Forum Infectious Diseases ◽

10.1093/ofid/ofaa439.518 ◽

2020 ◽

Vol 7 (Supplement_1) ◽

pp. S233-S234

Author(s):

Corrin Graue ◽

Bryan H Schmitt ◽

Amy Waggoner ◽

Frederic Laurent ◽

Lelia Abad ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Synovial Fluid ◽

Resistance Genes ◽

Clinical Care ◽

Joint Infection ◽

High Morbidity ◽

Bone And Joint Infection ◽

Microbiological Methods ◽

Antimicrobial Resistance Genes ◽

Wide Range

Abstract Background Bone and Joint Infections (BJIs) present with non-specific symptoms that may include pain, swelling, and fever and are associated with high morbidity and significant risk of mortality. BJIs can be caused by a variety of bacteria and fungi, including anaerobes and microorganisms that can be challenging to culture or identify by traditional microbiological methods. Clinicians primarily rely on culture to identify the pathogen(s) responsible for infection. The BioFire® Bone and Joint Infection (BJI) Panel (BioFire Diagnostics, Salt Lake City, UT) is designed to detect 15 gram-positive bacteria (including seven anaerobes), 14 gram-negative bacteria (including one anaerobe), two yeast, and eight antimicrobial resistance (AMR) genes from synovial fluid specimens in about an hour. The objective of this study was to evaluate the performance of an Investigational Use Only (IUO) version of the BioFire BJI Panel compared to various reference methods. Methods Remnant synovial fluid specimens, which were collected for routine clinical care at 13 study sites in the US and Europe, underwent testing using an IUO version of the BioFire BJI Panel. Performance of this test was determined by comparison to Standard of Care (SoC) consisting of bacterial culture performed at each study site according to their routine procedures. Results A total of 1544 synovial fluid specimens were collected and tested with the BioFire BJI Panel. The majority of specimens were from knee joints (77.9%) and arthrocentesis (79.4%) was the most common collection method. Compared to SoC culture, overall sensitivity was 90.2% and specificity was 99.8%. The BioFire BJI Panel yielded a total of 268 Detected results, whereas SoC yielded a total of 215 positive results for on-panel analytes. Conclusion The BioFire BJI Panel is a sensitive, specific, and robust test for rapid detection of a wide range of analytes in synovial fluid specimens. The number of microorganisms and resistance genes included in the BioFire BJI Panel, together with a reduced time-to-result and increased diagnostic yield compared to culture, is expected to aid in the timely diagnosis and appropriate management of BJIs. Disclosures Benjamin von Bredow, PhD, BioFire (Grant/Research Support) Jennifer Dien Bard, PhD, BioFire Diagnostic (Consultant, Scientific Research Study Investigator) Bart Kensinger, PhD, BioFire Diagnostics (Employee) Benedicte Pons, PhD, bioMerieux SA (Employee) Corinne Jay, PhD, bioMerieux SA (Employee)

Download Full-text

Metagenomic Quantification of Genes with Internal Standards

mBio ◽

10.1128/mbio.03173-20 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Emily Crossette ◽

Jordan Gumm ◽

Kathryn Langenfeld ◽

Lutgarde Raskin ◽

Melissa Duhaime ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Tetracycline Resistance ◽

Dairy Manure ◽

Molecular Techniques ◽

Gene Copy ◽

Gene Quantification ◽

Gene Copies ◽

Antimicrobial Resistance Genes ◽

Metagenomic Approach

ABSTRACT We demonstrate that an assembly-independent and spike-in facilitated metagenomic quantification approach can be used to screen and quantify over 2,000 genes simultaneously, while delivering absolute gene concentrations comparable to those for quantitative PCR (qPCR). DNA extracted from dairy manure slurry, digestate, and compost was spiked with genomic DNA from a marine bacterium and sequenced using the Illumina HiSeq4000. We compared gene copy concentrations, in gene copies per mass of sample, of five antimicrobial resistance genes (ARGs) generated with (i) our quantitative metagenomic approach, (ii) targeted qPCR, and (iii) a hybrid quantification approach involving metagenomics and qPCR-based 16S rRNA gene quantification. Although qPCR achieved lower quantification limits, the metagenomic method avoided biases caused by primer specificity inherent to qPCR-based methods and was able to detect orders of magnitude more genes than is possible with qPCR assays. We used the approach to simultaneously quantify ARGs in the Comprehensive Antimicrobial Resistance Database (CARD). We observed that the total abundance of tetracycline resistance genes was consistent across different stages of manure treatment on three farms, but different samples were dominated by different tetracycline resistance gene families. IMPORTANCE qPCR and metagenomics are central molecular techniques that have offered insights into biological processes for decades, from monitoring spatial and temporal gene dynamics to tracking ARGs or pathogens. Still needed is a tool that can quantify thousands of relevant genes in a sample as gene copies per sample mass or volume. We compare a quantitative metagenomic approach with traditional qPCR approaches in the quantification of ARG targets in dairy manure samples. By leveraging the benefits of nontargeted community genomics, we demonstrate high-throughput absolute gene quantification of all known ARG sequences in environmental samples.

Download Full-text

Antimicrobial Resistance and Virulence Determination of Enterococcus spp. Obtained from Hospital Environment in Iran

10.21203/rs.3.rs-123398/v1 ◽

2020 ◽

Author(s):

Saba Asgharzadeh Marghmalek ◽

Reza Valadan ◽

Mehrdad Gholami ◽

Mohtaram Nasrolahei ◽

Hamid Reza Goli

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Pathogenic Bacteria ◽

Antibiotic Resistance Genes ◽

Hospital Environment ◽

Diffusion Method ◽

Virulence Determinants ◽

Environmental Isolates ◽

Disk Diffusion Method ◽

Antimicrobial Resistance Genes

Abstract Background: The role of the hospital environment as a source of pathogenic bacteria in recent studies has been poorly investigated. This study investigated the distribution of antimicrobial resistance genes and virulence determinants in Enterococcus species isolated from hospital environment in Sari, Iran. Method: Overall, 90 enterococci strains were obtained from high touch surfaces of four hospitals in Sari, Iran. These environmental samples were obtained from bathroom, beds, tables, doorknobs, room keys, wheelchair and walls in the patient and staff’s rooms. The resistance profile of the isolates was determined by disk diffusion method. Seven resistance genes and two virulence associated genes were evaluated molecularly by multiplex PCR. Results: According to the PCR, 42 (46.66%) of them were E. faecalis and 48 (53.33%) others were detected as E. faecium. Also, 28 (66.6%) E. faecalis and 18 (37.5%) E. faecium isolates were multidrug-resistant (MDR). Among all 90 environmental isolates 54 (60%), 54 (60%), 8 (8.8%), 8 (8.8%), 60 (66.6%), 26 (28.8%), and 24 (26.6%) isolates contained tetM, tetL, vanA, vanB, ermB, aac(6´)-Ie-aph(2´´)-Ia, and aph (3´)-IIIa, respectively. Moreover, all isolates were investigated for the presence of virulence genes and 88 (97.7%) of isolates had esp gene, and 16 (17.7%) had ace.Conclusions: This report showed that the environmental isolates of Enterococcus are the major sources of antibiotic resistance genes that can transfer them to the clinical isolates of bacteria in hospital settings. An effective following strategy should be organized to clearance and stop emergence of these pathogenic bacteria.

Download Full-text

Detection and prevalence of antimicrobial resistance genes in Campylobacter spp. isolated from chickens and humans

Onderstepoort Journal of Veterinary Research ◽

10.4102/ojvr.v84i1.1411 ◽

2017 ◽

Vol 84 (1) ◽

Cited By ~ 13

Author(s):

Samantha Reddy ◽

Oliver T. Zishiri

Keyword(s):

Antibiotic Resistance ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

Pathogenic Bacteria ◽

Antibiotic Resistance Genes ◽

Quinolone Resistance ◽

Campylobacter Coli ◽

Antimicrobial Resistance Genes ◽

Campylobacter Spp ◽

Clinical Cases

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

Download Full-text

An omics-based framework for assessing the health risk of antimicrobial resistance genes

Nature Communications ◽

10.1038/s41467-021-25096-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

An-Ni Zhang ◽

Jeffry M. Gaston ◽

Chengzhen L. Dai ◽

Shijie Zhao ◽

Mathilde Poyet ◽

...

Keyword(s):

High Risk ◽

Antimicrobial Resistance ◽

Resistance Genes ◽

Fecal Microbiota Transplantation ◽

Antibiotic Resistance Genes ◽

Fecal Microbiota ◽

Clinical Applications ◽

World Health ◽

Antimicrobial Resistance Genes ◽

Health Organization

AbstractAntibiotic resistance genes (ARGs) are widespread among bacteria. However, not all ARGs pose serious threats to public health, highlighting the importance of identifying those that are high-risk. Here, we developed an ‘omics-based’ framework to evaluate ARG risk considering human-associated-enrichment, gene mobility, and host pathogenicity. Our framework classifies human-associated, mobile ARGs (3.6% of all ARGs) as the highest risk, which we further differentiate as ‘current threats’ (Rank I; 3%) - already present among pathogens - and ‘future threats’ (Rank II; 0.6%) - novel resistance emerging from non-pathogens. Our framework identified 73 ‘current threat’ ARG families. Of these, 35 were among the 37 high-risk ARGs proposed by the World Health Organization and other literature; the remaining 38 were significantly enriched in hospital plasmids. By evaluating all pathogen genomes released since framework construction, we confirmed that ARGs that recently transferred into pathogens were significantly enriched in Rank II (‘future threats’). Lastly, we applied the framework to gut microbiome genomes from fecal microbiota transplantation donors. We found that although ARGs were widespread (73% of genomes), only 8.9% of genomes contained high-risk ARGs. Our framework provides an easy-to-implement approach to identify current and future antimicrobial resistance threats, with potential clinical applications including reducing risk of microbiome-based interventions.

Download Full-text

First Report of Coexistence of blaSFO–1 and blaNDM–1 β-Lactamase Genes as Well as Colistin Resistance Gene mcr-9 in a Transferrable Plasmid of a Clinical Isolate of Enterobacter hormaechei

Frontiers in Microbiology ◽

10.3389/fmicb.2021.676113 ◽

2021 ◽

Vol 12 ◽

Author(s):

Wenxiu Ai ◽

Ying Zhou ◽

Bingjie Wang ◽

Qing Zhan ◽

Longhua Hu ◽

...

Keyword(s):

Antibiotic Resistance ◽

Antimicrobial Resistance ◽

Clinical Isolate ◽

Resistance Genes ◽

Antibiotic Resistance Genes ◽

Mobile Elements ◽

Conjugative Plasmid ◽

S1 Nuclease ◽

Antimicrobial Resistance Genes ◽

Enterobacter Hormaechei

Many antimicrobial resistance genes usually located on transferable plasmids are responsible for multiple antimicrobial resistance among multidrug-resistant (MDR) Gram-negative bacteria. The aim of this study is to characterize a carbapenemase-producing Enterobacter hormaechei 1575 isolate from the blood sample in a tertiary hospital in Wuhan, Hubei Province, China. Antimicrobial susceptibility test showed that 1575 was an MDR isolate. The whole genome sequencing (WGS) and comparative genomics were used to deeply analyze the molecular information of the 1575 and to explore the location and structure of antibiotic resistance genes. The three key resistance genes (blaSFO–1, blaNDM–1, and mcr-9) were verified by PCR, and the amplicons were subsequently sequenced. Moreover, the conjugation assay was also performed to determine the transferability of those resistance genes. Plasmid files were determined by the S1 nuclease pulsed-field gel electrophoresis (S1-PFGE). WGS revealed that p1575-1 plasmid was a conjugative plasmid that possessed the rare coexistence of blaSFO–1, blaNDM–1, and mcr-9 genes and complete conjugative systems. And p1575-1 belonged to the plasmid incompatibility group IncHI2 and multilocus sequence typing ST102. Meanwhile, the pMLST type of p1575-1 was IncHI2-ST1. Conjugation assay proved that the MDR p1575-1 plasmid could be transferred to other recipients. S1-PFGE confirmed the location of plasmid with molecular weight of 342,447 bp. All these three resistant genes were flanked by various mobile elements, indicating that the blaSFO–1, blaNDM–1, and mcr-9 could be transferred not only by the p1575-1 plasmid but also by these mobile elements. Taken together, we report for the first time the coexistence of blaSFO–1, blaNDM–1, and mcr-9 on a transferable plasmid in a MDR clinical isolate E. hormaechei, which indicates the possibility of horizontal transfer of antibiotic resistance genes.

Download Full-text

NanoARG: A web service for identification of antimicrobial resistance elements from nanopore-derived environmental metagenomes

10.1101/483248 ◽

2018 ◽

Author(s):

G. A. Arango-Argoty ◽

D. Dai ◽

A. Pruden ◽

P. Vikesland ◽

L. S. Heath ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistance Genes ◽

Sequence Data ◽

Antibiotic Resistance Genes ◽

Metal Resistance ◽

Direct Selection ◽

Human Pathogens ◽

Next Generation Sequencing Technology ◽

Monitoring Tools ◽

Long Reads

ABSTRACTDirect selection pressures imposed by antibiotics, indirect pressures by co-selective agents, and horizontal gene transfer are fundamental drivers of the evolution and spread of antibiotic resistance. Therefore, effective environmental monitoring tools should ideally capture not only antibiotic resistance genes (ARGs), but also mobile genetic elements (MGEs) and indicators of co-selective forces, such as metal resistance genes (MRGs). Further, a major challenge towards characterizing potential human risk is the ability to identify bacterial host organisms, especially human pathogens. Historically, short reads yielded by next-generation sequencing technology has hampered confidence in assemblies for achieving these purposes. Here we introduce NanoARG, an online computational resource that takes advantage of long reads produced by MinION nanopore sequencing. Specifically, long nanopore reads enable identification of ARGs in the context of relevant neighboring genes, providing relevant insight into mobility, co-selection, and pathogenicity. NanoARG allows users to upload sequence data online and provides various means to analyze and visualize the data, including quantitative and simultaneous profiling of ARG, MRG, MGE, and pathogens. NanoARG is publicly available and freely accessible at http://bench.cs.vt.edu/nanoARG.

Download Full-text

Antimicrobial Resistance and Virulence Determination of Enterococcus spp. isolated from Hospital Environment in Iran

10.21203/rs.3.rs-118272/v1 ◽

2020 ◽

Author(s):

Saba Asgharzadeh Marghmalek ◽

Reza Valadan ◽

Mehrdad Gholami ◽

Mohtaram Nasrolahei ◽

Hamid Reza Goli

Keyword(s):

Antimicrobial Resistance ◽

Resistance Genes ◽

Pathogenic Bacteria ◽

Antibiotic Resistance Genes ◽

Multidrug Resistant ◽

Hospital Environment ◽

Virulence Determinants ◽

Environmental Isolates ◽

Antimicrobial Resistance Genes ◽

Enterococcus Spp

Abstract Background: The role of the hospital environment as a source of pathogenic bacteria in recent studies has been poorly investigated. This study investigated the distribution of antimicrobial resistance genes and virulence determinants in Enterococcus species isolated from hospital environment in Sari, Iran. A total of 90 enterococci isolates were identified and species identification confirmed with specific primers. Seven resistance genes and two virulence associated genes were evaluated molecularly by multiplex polymerase chain reaction. Results: Of the 90 enterococcal isolates, 42 (46.66%), and 48 (53.33%) were identified as E. faecalis, and E. faecium, respectively. Also, 28 (66.6%) E. faecalis and 18 (37.5%) E. faecium isolates were multidrug-resistant (MDR). Among all 90 environmental isolates 54 (60%), 54 (60%), 8 (8.8%), 8 (8.8%), 60 (66.6%), 26 (28.8%), and 24 (26.6%) isolates contained tetM, tetL, vanA, vanB, ermB, aac (6´)-Ie-aph (2´´)-Ia, and aph (3´)-IIIa, respectively. Moreover, 88 (97.7%) and 16 (17.7%) isolates were detected as esp and ace positive ones, correspondingly. Conclusions: This report showed that the environmental isolates of Enterococcus are the major sources of antibiotic resistance genes that can transfer them to the clinical isolates of bacteria in hospital settings. An effective following strategy should be organized to clearance and stop emergence of these pathogenic bacteria.

Download Full-text

Draft Genome Sequence of Enterobacter hormaechei subsp. steigerwaltii Strain BEI01

Microbiology Resource Announcements ◽

10.1128/mra.00406-21 ◽

2021 ◽

Vol 10 (28) ◽

Author(s):

Soon Keong Wee ◽

Sharon Cui Mun Chan ◽

Xue Li Guan ◽

Eric Peng Huat Yap

Keyword(s):

Antimicrobial Resistance ◽

Genome Sequence ◽

Resistance Genes ◽

Draft Genome ◽

Gc Content ◽

Enterobacter Cloacae ◽

Metal Resistance ◽

Content Type ◽

Antimicrobial Resistance Genes ◽

Enterobacter Hormaechei

Here, we report the genome sequence of Enterobacter hormaechei subsp. steigerwaltii strain BEI01, originally deposited as a member of the Enterobacter cloacae complex. The genome is 4,900,246 bp in size with a GC content of 55.44%; it contains multidrug antimicrobial resistance genes and several metal resistance gene operons.

Download Full-text