Virulence gene profiles and phylogeny of Shiga toxin-positiveEscherichia colistrains isolated from FDA regulated foods during 2010-2017

Shiga Toxin ◽

In Silico ◽

Virulence Genes ◽

Virulence Gene ◽

Source Tracking ◽

Toxin Gene ◽

Food Testing ◽

AbstractIllnesses caused by Shiga toxin-producingEscherichiacoli (STECs) can be life threatening, such as hemolytic uremic syndrome (HUS). The STECs most frequently identified by USDA’s Microbiological Data Program (MDP) carried toxin gene subtypesstx1aand/orstx2a. Here we describe the genome sequences of 331 STECs isolated from foods regulated by the FDA 2010-2017, determining their genomic identity, serotype, sequence type, virulence potential, and prevalence of antimicrobial resistance. Isolates were selected from the MDP archive, routine food testing by field labs (ORA), food testing by a contract company, and our laboratory (ORS). Only 276 (83%) were confirmed as STECs byin silicoanalysis. Foods from which STECs were recovered included cilantro (6%), spinach (25%), lettuce (11%), and flour (9%). Phylogenetic analysis using core genome MLST revealed these STEC genomes were highly variable, with some clustering associated with ST types and serotypes. We detected 95 different sequence types (ST); several ST were previously associated with HUS: ST21 and ST29 (O26:H11), ST11 (O157:H7), ST33 (O91:H14), ST17 (O103:H2), and ST16 (O111:H-).in silicovirulome analyses showed ~ 51% of these strains were potentially pathogenic [besidesstxgene they also carriedeae(25%) or 26%subA(26%)]. Virulence gene prevalence was also determined:stx1 only (19%) -variants a and c;stx2 only (66%) – variants a, b, c, d, e, and g; andstx1/sxt2 (15%). Our data form a new WGS database that can be used to support food safety investigations and monitor the recurrence/emergence ofE. coliin foods.ImportanceShiga toxin-producingEscherichiacoli (STECs) are associated with foodborne outbreaks worldwide; however, surveillance has not previously included genomic analyses for phylogenetics, prevalence, or potential virulence. We constructed the first genomic database of isolates from FDA-regulated foods to help monitor the emergence of new pathogenic STECs. Although only ~30 STECs were isolated per year, 50% of these carried markers associated with pathogenesis either a combination ofeaeplusstx, orsubAplusstx. Moreover, those strains also carried virulence genes associated with severe illnesses. Here we showed that WGS enabled comparisons across isolates to establish phylogeny, help in identification of antibiotic resistance by monitoring the presence of antimicrobial resistance genes, and determined the presence of known virulence genes that have been linked with illnesses. Future food safety investigations will benefit from improved source tracking and risk assessments made possible by these analyses and new WGS database.

Virulence and antimicrobial resistance genes associated with the in-vivo pathogenicity of avian pathogenic E. coli isolates

GMPC Thesis and Opinions Platform ◽

10.51585/gtop.2021.0005 ◽

2021 ◽

Vol 1 (1) ◽

pp. 17-20

Author(s):

Ahmed Abd El-Mawgoud ◽

Azza El-Sawah ◽

Soad Nasef ◽

Al-Hussien Dahshan ◽

Ahmed Ali

Keyword(s):

Resistance Genes ◽

Broiler Chickens ◽

Virulence Genes ◽

Virulence Gene ◽

Health Concern ◽

Pathogenicity Test ◽

E Coli ◽

In the current study, ten avian pathogenic E. coli (APEC) isolates of the most predominant APEC serogroups isolated from broiler chickens in Egypt were screened for their virulence and antimicrobial resistance genes pattern using PCR. Five selected virulence gene patterns were further investigated for their in-vivo pathogenicity test. Results showed a 100% prevalence of the β-lactams and tetracyclines resistance genes. However, aminoglycoside and quinolone resistance genes were not detected. Also, 80% of the tested isolates harbored mcr-1 gene, colistin resistance gene. In-vivo pathogenic strains consistently harbored the virulence gene pattern of fimH, fimA, papC, iutA, and tsh. Additionally, the tsh gene was consistently detected with lethal APEC isolates in day-old chicks. These results highlighted the high prevalence of antimicrobial and virulence genes in APEC that potentially represent a public health concern. In this study, the virulence genes fimH, fimA, papC, iutA, and tsh were the most common virulence gene patterns associated with pathogenicity in day-old chicks.

Frequency and Characterization of Antimicrobial Resistance and Virulence Genes of Coagulase-Negative Staphylococci from Wild Birds in Spain. Detection of tst-Carrying S. sciuri Isolates

Microorganisms ◽

10.3390/microorganisms8091317 ◽

2020 ◽

Vol 8 (9) ◽

pp. 1317

Author(s):

Laura Ruiz-Ripa ◽

Paula Gómez ◽

Carla Andrea Alonso ◽

María Cruz Camacho ◽

Yolanda Ramiro ◽

...

Keyword(s):

Virulence Genes ◽

Meca Gene ◽

Virulence Gene ◽

Wild Birds ◽

Diffusion Method ◽

Coagulase Negative Staphylococci ◽

Disk Diffusion Method ◽

Resistance Phenotype ◽

The objective of this study was to determine the prevalence and diversity of coagulase-negative staphylococci (CoNS) species from wild birds in Spain, as well as to analyze the antimicrobial resistance phenotype/genotype and the virulence gene content. During 2015–2016, tracheal samples of 242 wild birds were collected in different regions of Spain for staphylococci recovery. The species identification was performed using MALDI-TOF. The antimicrobial resistance phenotype and genotype was investigated by the disk diffusion method and by PCR, respectively. The presence of the virulence genes lukF/S-PV, tst, eta, etb, etd and scn was investigated by PCR. Moreover, CoNS carrying the mecA gene were subjected to SCCmec typing. Of the tested animals, 60% were CoNS-carriers, and 173 CoNS isolates were recovered from the 146 positive animals, which belonged to 11 species, with predominance of S. sciuri (n = 118) and S. lentus (n = 25). A total of 34% of CoNS isolates showed a multidrug resistance phenotype, and 42 mecA-positive methicillin-resistant CoNS (MRCoNS) were detected. The isolates showed resistance to the following antimicrobials (percentage of resistant isolates/antimicrobial resistance genes detected): penicillin (49/ blaZ, mecA), cefoxitin (24/ mecA), erythromycin and/or clindamycin (92/ erm(B), erm(C), erm(43), msr(A), mph(C), lnu(A), lsa(B), vga(A) and sal(A)), gentamicin and/or tobramycin (5/ aac(6′)-Ie-aph(2″)-Ia, ant(4′)-Ia), streptomycin (12/str), tetracycline (17/ tet(K), tet(L), tet(M)), ciprofloxacin (4), chloramphenicol (1/ fexA), fusidic acid (86/ fusB, fusD) and trimethoprim–sulfamethoxazole (1/ dfrK). None of the isolates harbored the lukF/S-PV, eta, etb, etd and scn genes, but two S. sciuri isolates (1%) carried the tst gene. Wild birds are frequently colonized by CoNS species, especially S. sciuri. We identified scavenging on intensively produced livestock and feeding on landfills as risk factors for CoNS carriage. High proportions of MRCoNS and multidrug resistant CoNS were detected, which coupled with the presence of important virulence genes is of concern.

Shiga Toxin-Producing Escherichia coli (STEC) and STEC-Associated Virulence Genes in Raw Ground Pork in Canada

Journal of Food Protection ◽

10.4315/jfp-21-147 ◽

2021 ◽

Author(s):

Helen Zhang ◽

Etsuko Yamamoto ◽

Johanna Murphy ◽

Catherine Carrillo ◽

Annie Locas

Keyword(s):

Escherichia Coli ◽

Foodborne Pathogens ◽

Shiga Toxin ◽

Virulence Genes ◽

Virulence Gene ◽

Toxin Gene ◽

Gene Profiles ◽

Ground Pork ◽

Genes Encoding ◽

Human Illness

Shiga toxin-producing Escherichia coli (STEC) O157:H7/NM and some non-O157 STEC are foodborne pathogens. In response to pork-associated O157 STEC outbreaks in Canada, we investigated the occurrence of STEC in Canadian retail raw ground pork during the period of November 1, 2014 and March 31, 2016. Isolated STEC were characterized to determine the Shiga-toxin gene ( stx ) subtype and the presence of virulence genes encoding intimin ( eae ), and enterohemorrhagic E. coli hemolysin (hlyA) . O157 STEC and non-O157 STEC were isolated from 0.11% (1/879) and 2.24% (13/580) of the pork samples. STEC virulence gene profiles containing both eae and hlyA were found only in the O157 STEC ( stx 2a , eae , hlyA ) isolate. The eae gene was absent from all non-O157 STEC isolates. Of the 13 non-O157 STEC isolates, two virulence genes of stx 1a and hlyA were found in four (30.8%) O91:H14 STEC isolates, while one virulence gene of stx 2e, stx 1a , and stx 2a was identified in five (38.5%), two (15.4%) and one (7.7%) STEC isolates respectively of various serotypes. The remaining non-O157 STEC isolate carried stx 2 , but the subtype is unknown as this isolate could not be recovered for sequencing. O91:H14 STEC ( stx 1a, hlyA ) was previously reported in association with diarrhea illnesses, while the other non-O157 STEC isolates identified in this study are not known to be associated with severe human illnesses. Virulence gene profiles identified in this study indicate that the occurrence of non-O157 STEC capable of causing severe human illness is rare in Canadian retail pork. However, O157 STEC in ground pork can occasionally occur, therefore education regarding the potential risks associated with STEC contamination of pork would be beneficial for the public and those in the food industry in order to help reduce foodborne illnesses.

Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water

PLoS ONE ◽

10.1371/journal.pone.0245172 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245172

Author(s):

Meghan Maguire ◽

Julie A. Kase ◽

Dwayne Roberson ◽

Tim Muruvanda ◽

Eric W. Brown ◽

...

Keyword(s):

Escherichia Coli ◽

Food Safety ◽

Shiga Toxin ◽

Irrigation Water ◽

De Novo ◽

Limit Of Detection ◽

Bacterial Species ◽

Source Tracking ◽

Antimicrobial Resistance Genes ◽

Custom Script

Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Closed bacterial genomes from whole genome sequencing play an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using 24 hour enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore’s EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken2), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105−108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107−108 CFU/ml and a complete, fragmented MAG was obtained at 105−106 CFU/ml. In silico virulence detection for E. coli MAGs for 105−108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow provides proof of concept for faster detection and complete genomic characterization of STECs from a complex microbial sample compared to current reporting protocols and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.

Choice of library preparation and its effects on sequence quality, genome assembly, and precise in silico prediction of virulence genes in shiga toxin producing Escherichia coli

10.1101/2020.11.02.364646 ◽

2020 ◽

Author(s):

Julie Haendiges ◽

Karen Jinneman ◽

Narjol Gonzalez-Escalona

Keyword(s):

Escherichia Coli ◽

Illumina Sequencing ◽

Foodborne Pathogens ◽

Shiga Toxin ◽

Virulence Genes ◽

Sequence Data ◽

Virulence Gene ◽

Illumina Miseq ◽

Source Tracking ◽

Library Preparation

AbstractWhole genome sequencing (WGS) provides essential public health information and is used worldwide for pathogen surveillance, epidemiology, and source tracking. The sequencing of foodborne pathogens is commonly performed with Illumina sequencing chemistry to obtain data with high accuracy. The choice of library preparation method for highly complex organisms is very critical and can affect the final data output. The majority of Illumina sequencing platforms use rapid library preparation such as Nextera XT (transposon-based technology) (Illumina San Diego, CA), but this preparation has the potential to miss randomly distributed segments of genomes that might be important for downstream analyses. The Illumina Nextera DNA Prep library preparation kit, the successor of Nextera XT, shows better overall coverage of the complete genome. This study compared the quality of sequence data generated using Nextera XT and Nextera DNA Prep kits for DNA library preparation on an Illumina MiSeq, using a set of 30 O121:H19 shiga-toxin positive Escherichia coli strains isolated from flour during a 2016 outbreak. The performance of the two kits were evaluated using several metrics including sequencing quality, assembly quality, uniformity of genome coverage, and virulence gene identification. Overall, the results showed that in all of the analysed metrics, the Nextera DNA Prep kit performed outstanding in comparison to Nextera XT. The Nextera DNA Prep kit allowed for comprehensive detection of all virulence genes, which is of extremely high importance for making an educated assessment of the virulence potential of Escherichia coli. This comprehensive side-by-side comparison will be of significance for those interested in improving their sequencing workflow for STECs and the determination of health risks using WGS data.

Molecular characterization of antimicrobial resistance and virulence genes of Escherichia coli isolates from bovine mastitis

Veterinary World ◽

10.14202/vetworld.2020.1588-1593 ◽

2020 ◽

Vol 13 (8) ◽

pp. 1588-1593

Author(s):

Zuhair Bani Ismail ◽

Sameeh M. Abutarbush

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Virulence Genes ◽

Bovine Mastitis ◽

Virulence Gene ◽

Diffusion Method ◽

Disk Diffusion Method ◽

E Coli ◽

Procaine Penicillin

Background and Aim: Mastitis is a common and economically important disease in dairy cattle. It remains one of the most common reasons for the extensive use of antimicrobials in dairy farms leading to the emergence of antimicrobial-resistant pathogens. The aim of this study was to determine the patterns of antimicrobial resistance of Escherichia coli isolates from bovine mastitis and to identify prominent antimicrobial resistance and virulence genes among isolated strains. Materials and Methods: Antimicrobial susceptibility testing against six antibiotic groups, including tetracyclines, aminoglycosides, beta-lactams, macrolides, sulfonamides, and fluoroquinolones was performed using the disk diffusion method. PCR was performed on resistant isolates to detect resistance and virulence genes using commercially available primers. Results: Out of 216 milk samples cultured, 14 samples yielded E. coli isolates. All isolates (100%) were resistant to ampicillin, amoxicillin, procaine penicillin, streptomycin, oxytetracycline, and sulfamethoxazole-trimethoprim. Only one isolate (7%) was sensitive to gentamicin, and all isolates (100%) were sensitive to enrofloxacin and ciprofloxacin. All isolates carried at least one resistance gene against one or more of the major antibiotic groups. All isolates carried the ereA, tetG, tetE, and tetB genes, followed by tetA (93%), ampC (86%), strA (86%), sul1 (78%), tetD (71%), tetC (57%), aadA (57%), and strB (36%). The lowest percentage of isolates carried bla1 (17%) and bla2 (12%) genes, and none of the isolates carried the qnrA gene. Most of the isolates (93%) carried the Shiga toxin 1 virulence gene, followed by complement resistance protein (79%), intimin (64%), Shiga toxin 2 (36%), cytotoxic necrotizing factor (35%), aerotaxis receptor (21%), and type 1 fimbriae (15%). Conclusion: Results of this study indicate that the high percentages of E. coli isolate from bovine mastitis are resistant to two or more of the major antibiotic groups, irrespective of the presence or absence of relevant resistance or virulence genes.

Culture-Independent Genotyping, Virulence and Antimicrobial Resistance Gene Identification of Staphylococcus aureus from Orthopaedic Implant-Associated Infections

Microorganisms ◽

10.3390/microorganisms9040707 ◽

2021 ◽

Vol 9 (4) ◽

pp. 707

Author(s):

J. Christopher Noone ◽

Fabienne Antunes Ferreira ◽

Hege Vangstein Aamot

Keyword(s):

Staphylococcus Aureus ◽

Resistance Genes ◽

Virulence Gene ◽

Orthopaedic Implant ◽

Metagenomic Sequencing ◽

Gene Detection ◽

Shotgun Metagenomic Sequencing ◽

Antimicrobial Resistance Genes ◽

Culture Independent

Our culture-independent nanopore shotgun metagenomic sequencing protocol on biopsies has the potential for same-day diagnostics of orthopaedic implant-associated infections (OIAI). As OIAI are frequently caused by Staphylococcus aureus, we included S. aureus genotyping and virulence gene detection to exploit the protocol to its fullest. The aim was to evaluate S. aureus genotyping, virulence and antimicrobial resistance genes detection using the shotgun metagenomic sequencing protocol. This proof of concept study included six patients with S. aureus-associated OIAI at Akershus University Hospital, Norway. Five tissue biopsies from each patient were divided in two: (1) conventional microbiological diagnostics and genotyping, and whole genome sequencing (WGS) of S. aureus isolates; (2) shotgun metagenomic sequencing of DNA from the biopsies. Consensus sequences were analysed using spaTyper, MLST, VirulenceFinder, and ResFinder from the Center for Genomic Epidemiology (CGE). MLST was also compared using krocus. All spa-types, one CGE and four krocus MLST results matched Sanger sequencing results. Virulence gene detection matched between WGS and shotgun metagenomic sequencing. ResFinder results corresponded to resistance phenotype. S. aureus spa-typing, and identification of virulence and antimicrobial resistance genes are possible using our shotgun metagenomics protocol. MLST requires further optimization. The protocol has potential application to other species and infection types.

Carbapenem-Resistant Pseudomonas aeruginosa Strains-Distribution of the Essential Enzymatic Virulence Factors Genes

Antibiotics ◽

10.3390/antibiotics10010008 ◽

2020 ◽

Vol 10 (1) ◽

pp. 8

Author(s):

Tomasz Bogiel ◽

Małgorzata Prażyńska ◽

Joanna Kwiecińska-Piróg ◽

Agnieszka Mikucka ◽

Eugenia Gospodarek-Komkowska

Keyword(s):

Pseudomonas Aeruginosa ◽

Alkaline Protease ◽

Virulence Genes ◽

Virulence Gene ◽

Hospital Environment ◽

Gene Encoding ◽

Virulence Gene Expression ◽

Carbapenem Resistant ◽

Antimicrobial Resistance Genes ◽

The Difference

Pseudomonas aeruginosa is one of the most commonly isolated bacteria from clinical specimens, with increasing isolation frequency in nosocomial infections. Herein, we investigated whether antimicrobial-resistant P. aeruginosa strains, e.g., metallo-beta-lactamase (MBL)-producing isolates, may possess a reduced number of virulence genes, resulting from appropriate genome management to adapt to a changing hospital environment. Hospital conditions, such as selective pressure, may lead to the replacement of virulence genes by antimicrobial resistance genes that are crucial to survive under current conditions. The study aimed to compare, using PCR, the frequency of the chosen enzymatic virulence factor genes (alkaline protease-aprA, elastase B-lasB, neuraminidases-nan1 and nan2, and both variants of phospholipase C-plcH and plcN) to MBL distribution among 107 non-duplicated carbapenem-resistant P. aeruginosa isolates. The gene encoding alkaline protease was noted with the highest frequency (100%), while the neuraminidase-1 gene was observed in 37.4% of the examined strains. The difference in lasB and nan1 prevalence amongst the MBL-positive and MBL-negative strains, was statistically significant. Although P. aeruginosa virulence is generally more likely determined by the complex regulation of the virulence gene expression, herein, we found differences in the prevalence of various virulence genes in MBL-producers.

Serotype distribution, antimicrobial susceptibility, antimicrobial resistance genes and virulence genes of Salmonella isolated from a pig slaughterhouse in Yangzhou, China

AMB Express ◽

10.1186/s13568-019-0936-9 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Quan Li ◽

Jian Yin ◽

Zheng Li ◽

Zewei Li ◽

Yuanzhao Du ◽

...

Keyword(s):

Public Health ◽

Resistance Genes ◽

Antimicrobial Susceptibility ◽

Virulence Genes ◽

Multidrug Resistant ◽

Resistance Rate ◽

Economic Losses ◽

Production Chain ◽

AbstractSalmonella is an important food-borne pathogen associated with public health and high economic losses. To investigate the prevalence and the characteristics of Salmonella in a pig slaughterhouse in Yangzhou, a total of 80 Salmonella isolates were isolated from 459 (17.43%) samples in 2016–2017. S. Derby (35/80, 43.75%) was the most prevalent, followed by S. Rissen (16/80, 20.00%) and S. Newlands (11/80, 13.75%). The highest rates of susceptibility were observed to cefoxitin (80/80, 100.0%) and amikacin (80/80, 100.0%), followed by aztreonam (79/80, 98.75%) and nitrofurantoin (79/80, 98.75%). The highest resistance rate was detected for tetracycline (65/80, 81.25%), followed by ampicillin (60/80, 75.00%), bactrim (55/80, 68.75%), and sulfisoxazole (54/80, 67.50%). Overall, 91.25% (73/80) of the isolates were resistant to at least one antibiotic, while 71.25% (57/80) of the isolate strains were multidrug resistant in the antimicrobial susceptibility tested. In addition, 86.36% (19/22) of the 22 antimicrobial resistance genes in the isolates were identified. Our data indicated that the resistance to certain antimicrobials was significantly associated, in part, with antimicrobial resistance genes. Furthermore, 81.25% (65/80) isolates harbored the virulence gene of mogA, of which 2 Salmonella Typhimurium isolates carried the mogA, spvB and spvC virulence genes at the same time. The results showed that swine products in the slaughterhouse were contaminated with multidrug resistant Salmonella commonly, especially some isolates carry the spv virulence genes. The virulence genes might facilitate the dissemination of the resistance genes to consumers along the production chain, suggesting the importance of controlling Salmonella during slaughter for public health.