Scholarly article on topic 'Isolation, genotyping and antimicrobial resistance of Shiga toxin-producing  Escherichia coli'

Isolation, genotyping and antimicrobial resistance of Shiga toxin-producing Escherichia coli Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Bianca A. Amézquita-López, Marcela Soto-Beltrán, Bertram G. Lee, Jaszemyn C. Yambao, Beatriz Quiñones

Abstract Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen linked to outbreaks of human gastroenteritis with diverse clinical spectra. In this review, we have examined the currently methodologies and molecular characterization techniques for assessing the phenotypic, genotypic and functional characteristics of STEC O157 and non-O157. In particular, traditional culture and isolation methods, including selective enrichment and differential plating, have enabled the effective recovery of STEC. Following recovery, immunological serotyping of somatic surface antigens (O-antigens) and flagellum (H-antigens) are employed for the classification of the STEC isolates. Molecular genotyping methods, including multiple-locus variable-number tandem repeat analysis, arrays, and whole genome sequencing, can discriminate the isolate virulence profile beyond the serotype level. Virulence profiling is focused on the identification of chromosomal and plasmid genes coding for adhesins, cytotoxins, effectors, and hemolysins to better assess the pathogenic potential of the recovered STEC isolates. Important animal reservoirs are cattle and other small domestic ruminants. STEC can also be recovered from other carriers, such as mammals, birds, fish, amphibians, shellfish and insects. Finally, antimicrobial resistance in STEC is a matter of growing concern, supporting the need to monitor the use of these agents by private, public and agricultural sectors. Certain antimicrobials can induce Shiga toxin production and thus promote the onset of severe disease symptoms in humans. Together, this information will provide a better understanding of risks associated with STEC and will aid in the development of efficient and targeted intervention strategies.

Academic research paper on topic "Isolation, genotyping and antimicrobial resistance of Shiga toxin-producing Escherichia coli"

Journal of Microbiology, Immunology and Infection (2017) xx, 1-10

Available online at www.sciencedirect.com

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journal homepage: www.e-jmii.com

Review Article

isolation, genotyping and antimicrobial resistance of Shiga toxin-producing Escherichia coli

Bianca A. Amézquita-López a *, Marcela Soto-Beltran a, Bertram G. Lee b, Jaszemyn C. Yambao b, Beatriz Quiñones b **

a Universidad Autónoma de Sinaloa, Facultad de Ciencias Qu ímico Biológicas, Culiacan, Sinaloa, Mexico b United States Department of Agriculture/Agricultural Research Service, Western Regional Research Center, Produce Safety and Microbiology Research Unit, Albany, CA, USA

Received 11 December 2016; received in revised form 28 June 2017; accepted 12 July 2017 Available online ■ ■ ■

KEYWORDS

Antimicrobials; Escherichia coli; Food safety; Genotyping; Zoonosis

Abstract Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen linked to outbreaks of human gastroenteritis with diverse clinical spectra. In this review, we have examined the currently methodologies and molecular characterization techniques for assessing the phenotypic, genotypic and functional characteristics of STEC O157and non-O157. In particular, traditional culture and isolation methods, including selective enrichment and differential plating, have enabled the effective recovery of STEC. Following recovery, immunological serotyping of somatic surface antigens (O-antigens) and flagellum (H-antigens) are employed for the classification of the STEC isolates. Molecular genotyping methods, including multiple-locus variable-number tandem repeat analysis, arrays, and whole genome sequencing, can discriminate the isolate virulence profile beyond the serotype level. Virulence profiling is focused on the identification of chromosomal and plasmid genes coding for adhesins, cytotoxins, effectors, and hemolysins to better assess the pathogenic potential of the recovered STEC isolates. Important animal reservoirs are cattle and other small domestic ruminants. STEC can also be recovered from other carriers, such as mammals, birds, fish, amphibians, shellfish and insects. Finally, antimicrobial resistance in STEC is a matter of growing concern, supporting the need to monitor the use of these agents by private, public and agricultural sectors. Certain antimicrobials can induce Shiga toxin production and thus promote the onset of severe disease

* Corresponding author. Universidad Autónoma de Sinaloa, Facultad de Ciencias Químico Biológicas, Ciudad Universitaria, Av. Las Americas & Josefa Ortiz de Domínguez, Culiacán Rosales, CP 80013, Sinaloa, Mexico. Fax: +52 (667)713 7860.

** Corresponding author. U.S. Department of Agriculture/ARS, Western Regional Research Center, Produce Safety and Microbiology Research Unit, 800 Buchanan Street, Albany, CA, 94710, USA. Fax: +1 510 559 6162.

E-mail addresses: bamezquita@uas.edu.mx (B.A. Amázquita-Lopez), Beatriz.Quinones@ars.usda.gov (B. Quinones).

http://dx.doi.org/10.1016/j.jmii.2017.07.004

1684-1182/Copyright © 2017, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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2 B.A. Amézquita-López et al.

symptoms in humans. Together, this information will provide a better understanding of risks associated with STEC and will aid in the development of efficient and targeted intervention strategies.

Copyright © 2017, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen that have been linked to outbreaks from food-borne and waterborne sources. STEC causes human gastrointestinal illnesses with diverse clinical spectra, ranging from watery and bloody diarrhea to hemorrhagic colitis.1-4 In some rare cases, infection can result in the life-threatening, hemolytic uremic syndrome (HUS), and it is thought that Shiga toxins (Stx) are the key virulence factors contributing to the development of HUS.1'3'4 Although more than 400 different serotypes of STEC have been isolated, O157:H7 is the serotype that has been most studied since it has been commonly associated with the development of severe human illness.5 Recent epidemiological studies have revealed other STEC non-O157 serotypes, O26:H2, O45:H2, O103:H11, O111:H8, O121:H19, and O145:H28, to be highly associated with human disease.2

Due to the clinical importance of STEC in recent years, a number of methods have been developed to determine the diversity, virulence, and phylogenetic relationships of STEC isolates. These methods have enabled the monitoring of STEC outbreaks and traceback investigations of contamination sources.6,7 The objective of this review article is to examine current knowledge of techniques for the pheno-typic and genotypic characterization of STEC O157 and non-O157. This information will provide a better understanding of risks associated with STEC and will aid in the development of efficient and targeted intervention strategies.

Routes of transmission and mechanisms of pathogenicity in humans

STEC infections are usually acquired by ingestion of contaminated food, water, or by contact from person to person (Fig. 1).1 A large portion of STEC infections have been attributed to the consumption of undercooked contaminated food, usually meat and dairy products.8,9 In particular, ground meat is considered a common transmission vehicle of STEC due to the ease of cross-contamination during preparation. Also, the uneven dispersion of STEC throughout the substrate results in an inefficient killing of this pathogen in ground beef after heat exposure during cooking.8 Certain "super-shedding" animals, which are considered main STEC reservoirs, can excrete high concentration levels of STEC in feces and are also an important source of human infections and environmental contamination (Fig. 1).1,8,10 Consequently, the dispersed pathogen can then attach to a variety of fruits and vegetables depending on the species and specific

conditions.8 Infection can also occur from swimming, drinking or bathing with contaminated water or occupying grazing areas presumably strewn with manure. Human infections have been attributed to direct contact with dogs, sheep, horses and goats at petting zoos, open farms and animal shows (Fig. 1).8,10 Person-to-person or secondary transmission is important in propagation of outbreaks and can account for 15—20% of the cases.

The mechanism of pathogenicity is mainly attributed to the production of Stx. Infection begins once Stx bind to the cell-surface receptor on the endothelial cells. Thereafter, the catalytic A-subunit is translocated into the cell cytosol, resulting in the inhibition of protein synthesis after inacti-vation of 60S ribosomal subunit of the eukaryotic cell.4,11 STEC infections require a low infectious dose (<50 bacterial cells),1,4,12 and the incubation period, prior to the onset of diarrhea, ranges between 2 and 12 days.11 Typical initial symptoms include abdominal pain, diarrhea, fever and vomiting, followed by bloody diarrhea in about 90% of the cases.11 Bacteremia is almost never found in conjunction with an enteric STEC infection. Instead, systemic complications associated with HUS arise from lesions caused by circulating Stx as soluble free Stx or by binding to blood components such as leukocytes, monocytes or red blood cells.4,11 The rate at which severe disease symptoms result in HUS varies widely (0—15%), and death due to HUS occurs in approximately 5% of the patient.

Animal reservoirs

Studies of zoonotic STEC have shown that cattle is considered the main reservoir for STEC strains,1,8—10 and over 430 STEC serotypes have been detected in isolates recovered from cattle.9 Other domestic small ruminants, such as sheep and goats are also important carriers of STEC especially outside of the United States.10,13,14 In particular, sheep and their products have been documented as reservoirs of a diverse set of non-O157 serotypes STEC (O26, O91, O115, O128, and 0130), encoding key virulence factors that have been implicated in human disease and are important reservoirs in Australia and Norway.10,13,14 Water buffalo is an important reservoir of STEC O157 in countries in Asia, South America and Europe. In Bangladesh, STEC was isolated from 38% of buffaloes sampled before slaughter, and in Vietnam 28% of the animals surveyed were STEC positive although they were not O157.10

STEC has also been identified in a wide variety of other carriers, including mammals, birds, amphibians, fish, shellfish and insects.1,8,10,14,15 There is some evidence that non-ruminants may be categorized as spillover hosts; these hosts do not maintain STEC levels without continual

STEC detection and characterization

Figure 1. Transmission routes of STEC infections. STEC infections are usually acquired by ingestion of contaminated food or water. Certain animal reservoirs can excrete STEC in high concentrations which may subsequently contribute to the contamination of produce in agricultural fields, when animal manure is used as fertilizer. Contaminated water used for swimming, drinking, bathing or irrigation of agricultural fields are also relevant sources of STEC infection. Human STEC infections have also been attributed to direct contact with domestic farm animals at petting zoos, open farms and animal shows.

exposure to STEC. However, spillover hosts may still spread STEC over a wide area especially in the case of migratory birds traveling a long distance in a single day.10 Some shellfish and other aquatic species may act as dead-end hosts for STEC since they only transmit STEC when they are consumed.10 Most animal hosts are asymptomatic because they lack vascular receptors for Stx with pigs being a notable exception.

Isolation and culture

Culturing and isolation methods are generally regarded as the standard procedure for pathogen detection. These traditional recovery methods consist of several steps, enrichment, followed by selective and differential plating for isolation, and then by serological or molecular tests for confirmation (Fig. 2).16,17 Enrichment facilitates the resuscitation of bacteria exposed to stress or growth inhibitors in the tested matrix and enables the recovery of isolates when present at low concentration in the tested sample.7,12,16,17 Common growth media used for the enrichment of STEC O157:H7 and non-O157 include tryptic soy broth, E. coli broth and buffered peptone water (Fig. 2). Typical incubation conditions are 35—37 °C for a

period of 16—24 h. To suppress a potential antagonistic activity of competing microflora, the sample can be incubated at a higher temperature of 42 °C; however, the use of this temperature may also interfere with the recovery of damaged STEC cells.15,18 Selective enrichment broths are often supplemented with agents such as bile salts, potassium tellurite and novobiocin, but it has been reported that the addition of novobiocin can inhibit the growth of some STEC isolates.7,12 Moreover, it has been demonstrated that avoiding the use of antibiotics in enrichment may help to increase the number of STEC recovered from a complex sample.13,16,18,19

Unlike commensal E. coli, typical E. coli O157:H7 do not ferment sorbitol and lack the ability to produce b-D-glucu-ronidase. Sorbitol-containing MacConkey agar (Difco Labs, Detroit, MI, USA), supplemented with cefixime and tellurite, has been effective in the isolation of STEC O157.20 The use of chromogenic agars such as Rainbow® O157 Agar (Biolog, Hayward, CA, USA) or CHROMagar™ (CHROMagar, Paris, France) are suitable for screening major STEC O-an-tigen serogroups associated with human illness (Fig. 2).7,15,21 Following enrichment and prior to plating on selective media, immunomagnetic separation (IMS), using antibodies for the specific recognition of E. coli O-antigen serogroups, is employed as a concentration step (Fig. 2).

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Figure 2. Culture and isolation methods for efficient STEC recovery. Recovery methods for STEC consist of several steps such as enrichment, separation, followed by the selective and differential plating for colony isolation. Common growth liquid media are used for the enrichment broth step, enabling the resuscitation of stressed or injured cells. A key step in STEC recovery involves the immunomagnetic separation prior to plating on various selective chromogenic media, resulting in the isolation of STEC colonies with a distinctive color morphology.

IMS has been demonstrated to provide a greater sensitivity and reliability in the recovery of E. coli O157:H7 and non-0157 from animal fecal samples.13,15,18,22

STEC classification schemes

One classification scheme that has been traditionally used for categorizing STEC is immunological serotyping (Table 1).23,24 STEC serotyping is based on combinations of 174 somatic surface antigens (O-antigens) and 53 flagellum (H-antigens).23,24 Over 400 different serotypes of STEC have been identified and several serotypes have been further classified into seropathotypes, based on reported frequency and severity of illness.5 These designations range from seropathotype A for relatively high incidence and association with severe disease to seropathotype E for no illness in humans.5 Serotypes belonging to seropathotype A are the most virulent and include O157:H7 and O157:NM (NM; not mobile). Seropathotype B comprises serotypes O126:H11, O103:H2, O111:NM, O121:H19, and O145:NM, which have been associated with severe disease symptoms (HUS) but less frequently than serotype O157. Seropathotype C is composed of serotypes O91:H21 and O113:H21, which are both associated with outbreaks but rarely

implicated in causing HUS. Seropathotype D represents serotypes that have been implicated in sporadic cases of diarrhea; and seropathotype E contains all STEC serotypes that have not been linked to human diseases.5

Molecular methods have recently emerged for discriminating or fingerprinting beyond the level of the STEC serotype and also for determining the STEC isolate relatedness and attributing sources of contamination (Table 1). Each method has different advantages and limitations, and the selection of the best method to use will be dependent upon the level of the typing resolution desired by the end user. One of these STEC classification methods consists of subtyping by pulsed-field gel electrophoresis (PFGE), which has a high power of discrimination as well as reproducibility and ease of standardization.25 PFGE uses restriction enzymes to generate DNA fragments in sizes spanning the entire genome.25 The most-commonly used restriction enzyme for digestion of the STEC genome is Xbal, which recognizes a rare sequence in bacterial genomes with more than 45% of GC content. Using a second restriction enzyme leads to a better discrimination of identical PFGE patterns, when bacterial isolates are suspected to be epidemiologi-cally linked and when investigations of large-scale outbreaks are needed.26 Several studies suggest that combining PFGE results with data obtained with other

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Table 1 Summary of methods to characterize, fingerprint and genotype STEC.

Method

Description of technique

Purpose

Advantage

Disadvantage

Immunological-based method

Nucleic acid-based methods

Serotyping

Polymerase chain reaction (PCR)

Real-time PCR

Pulsed-field gel electrophoresis (PFGE)

Multilocus VNTR analysis (MLVA)

DNA microarray

Whole genome sequencing (WGS)

Uses specific antisera to identify O- and H-antigens.

Amplifies target using sequence-specific primers.

Uses fluorophore-labeled probe to detect specific amplification of target sequence.

Uses restriction enzymes to fragment the entire genome and separates large size fragments on an agarose gel with pulsed-field electrophoresis, which continually changes the direction of the electrical current.

Measures number of copies of repeats at different short regions of repeated DNA sequences, known as variable number tandem repeat (VNTR). Detects complementary nucleotide sequences in tested bacterial isolates by measuring hybridization to DNA probes attached in an ordered fashion to a solid support. Generates multiple short sequence reads across the entire genome and then assemble them based on overlapping regions among the reads.

Serotype characterization

Detection/

virulence

characterization

Detection/

virulence

characterization

Fingerprinting

Fingerprinting

Virulence

characterization/

genotyping

Virulence

characterization/

genotyping

Standard method used by laboratories for species classifications.

Simple, rapid and cost effective typing method.

Faster than regular PCR; quantification of target is possible.

Good resolution and considered a standard method.

Antisera cross-reactivity of some can lead to false positive results; time consuming; immunological reagents can be limited in amounts.

Needs extra time to for results analysis on agarose gel.

Requires expensive instrumentation and reagents for real-time detection relative to conventional PCR. May not be able to discriminate among some clonal bacterial strains.

Rapid and high throughput; allows the discrimination of certain strains not distinguished by PFGE.

Simultaneously screen for multiple markers in a large number of samples; can detect single nucleotide polymorphisms.

Enhanced resolution with best strain discrimination; enables the detection of unknown single nucleotide polymorphisms.

Requires expensive instrumentation and fluorophore-conjugated reagents.

High cost of specialized and non-portable fluorescent array scanners. Colorimetric detection assays use unstable reagents, leading to overexposure. Substantial cost in time and instrumentation. Requires large computational servers. In silico data comparison has to be standardized.

Table 2 Characteristics and/or associated functions of virulence genes.

Target gene Location Characteristics and/or associated functions

Hemolysins

ehxA po157 plasmid Enterohemolysin; produces small turbid zones of lysed red blood cells

hlyA Chromosome a-hemolysin; produces large clear zones of lysed red blood cells

sheA Chromosome induced hemolysin; found in pathogenic and non-pathogenic E. coli

Adhesins

eae LEE region Intimin; forms attaching and effacing lesions

saa po113 plasmid STEC autoagglutinating adhesin; associated with non-O157 LEE-negative

Effectors

ent/espL2 o-Island 122 Effector; alters cytoskeleton in human cells

espK Prophage Sp6 Effector; unknown function

espN Prophage Sp6 Effector; unknown function

espP po157 plasmid Extracellular serine protease

katP po157 plasmid EHEC catalase-peroxidase

nleA o-Island 71 Effector; dirupts protein secretion

nleB o-Island 122 Effector; interfers with inflammatory signaling pathways

nleE o-Island 122 Effector; interfers with inflammatory signaling pathways

nleH1-2 o-Island 71 Effector; interfers with inflammatory signaling pathways

Cytotoxins

stx1a Chromosome Stx1 prototype; 1000 times less cytotoxic than Stx2a, repressed by iron

stx1c Chromosome Stx variant linked to mild symptoms in humans; common in ovine STEC

stx1d Chromosome Stx variant not associated with a particular food source

stx2a Chromosome Stx2 prototype; linked to severe HUS in humans

stx2b Chromosome Stx variant linked to eae-negative STEC and mild disease in humans

stx2c Chromosome Stx variant linked to diarrhea and HUS in humans

stx2d Chromosome Stx variant found in highly virulent strains; Stx activity increased by elastase

stx2e Chromosome Stx variant responsible for edema in pigs; rare in human disease

stx2f Chromosome Stx variant isolated from pigeon; rare in human disease

stx2g Chromosome Stx variant common in bovine STEC

subA po113 plasmid Subtilase cytotoxin; triggers apoptosis in human cells

O-antigens

wzyo26 Chromosome E. coli O26 O-antigen polymerase

wzyO45 Chromosome E. coli O45 O-antigen polymerase

wzxo91 Chromosome E. coli O91 O-antigen flippase

wzyo103 Chromosome E. coli O103 O-antigen polymerase

wzy0104 Chromosome E. coli O104 O-antigen polymerase

wzyom Chromosome E. coli O111 O-antigen polymerase

wzyo113 Chromosome E. coli O113 O-antigen polymerase

wzyo121 Chromosome E. coli O121 O-antigen polymerase

wzyo128 Chromosome E. coli O128 O-antigen polymerase

wzyo145 Chromosome E. coli O145 O-antigen polymerase

wzyo157 Chromosome E. coli O157 O-antigen polymerase

H-antigens

fliCH2 Chromosome E. coli flagellar H2 antigen

fliCH7 Chromosome E. coli flagellar H7 antigen

fliCH8 Chromosome E. coli flagellar H8 antigen

fliCun Chromosome E. coli flagellar H11 antigen

fliCH19 Chromosome E. coli flagellar H19 antigen

fliCH21 Chromosome E. coli flagellar H21 antigen

subtyping techniques helps to increase the discriminative power of PFGE.13,18,27,28

Another genotyping method that has been developed to discriminate STEC from multiple sources is multiple locus variable-number tandem repeat analysis (MLVA) (Table 1).13,18,27,28 MLVA amplifies short regions of repeated DNA sequences, known as variable number tandem repeat (VNTR), differing in size, location and number of copies.28

When selecting VNTR loci for MLVA, the stability of the locus is important for results interpretation. The advantage of using MLVA as a typing method is that it is rapid and high throughput and allows the discrimination of certain strains by non-typeable PFGE.13,27,28 In recent years, MLVA has become less expensive and more accessible, and has resulted in the implementation of this typing scheme for monitoring pathogen surveillance.7

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Table 3 Summary of antimicrobial resistance in STEC, as described in previous reports.19,43 49

Antimicrobial class Antimicrobial agent Function inhibited Domestic animal host

Aminoglycosides Amikacin Bacterial protein synthesis Cattle, Sheep

Gentamicin Sheep

Kanamycin Cattle, Sheep

ß-Lactamase inhibitors Amoxicillin — Cell wall synthesis; some ß-lactamases Cattle

Clavulanic acid

Cephems (parenteral) Cephalothin Cell wall synthesis Cattle, Sheep

Cefoperazone Cattle

Ceftazidime Cattle

Ceftriaxone Cattle

Folate pathway inhibitors Trimethoprim- Folic acid synthesis Cattle, Chicken, Pig, Turkey

Sulfamethoxazole

Fosfomycins Fosfomycin Enzymes involved in cell wall synthesis Cattle, Pig

Lipopeptides Colistin Bacterial membrane permeability Cattle, Chicken, Pig, Turkey

Macrolides Erythromycin Bacterial protein synthesis Pig

Penems Imipenem Cell wall synthesis Sheep

Penicillins Ampicillin Cell wall synthesis Cattle, Chicken, Sheep

Phenicols Chloramphenicol RNA synthesis Cattle, Sheep

Quinolones Ciprofloxacin DNA synthesis Cattle, Turkey

Nalidixic acid Cattle, Chicken, Pig, Turkey

Tetracyclines Tetracycline Bacterial protein synthesis Pig, Sheep

Polymerase chain reaction (PCR) assays are the preferred method by most research and surveillance laboratories for routine analysis since it is cost effective and simple. In particular, real-time PCR assays can yield faster results but are limited in the number of targets to be analyzed and also require expensive instrumentation and assay reagents (Table 1). Given the limitations of available detection methods, improvements in the cost-effectiveness and reliability of procedures are still required for routine high-throughput pathogen surveillance. Other molecular-based genotyping technologies, such as DNA microarrays, can simultaneously screen multiple set of specific markers for categorizing STEC in a large number of samples (Table 1).6,29 Array-based detection is still less expensive than new sequencing technologies and is not subject to the challenging analysis of massive amounts of data when compared to sequencing.30 Some microarray methods have been used for identifying STEC.6,31-33 However, some of the available array methods, using fluorescent assays, can result in reduced sensitivities.34 Alternative methods, using novel colorimetric technology, have been developed for genotyping of STEC O157 and non-O157 from livestock and wildlife in major agricultural regions in the United States.15,31,33

Whole genome sequencing is being used increasingly in strain typing since this method provides an enhanced resolution when compared to other genotyping methods (Table 1).7,35 The advantage of genome sequencing is that it allows the examination of the entire genome instead of just specific genomic regions or markers and enables the detection of unknown single nucleotide polymorphisms. Sequence reads of genomic fragments are either analyzed directly or assembled into contigs to form a draft genome. Once assembled, genome comparisons lead to a better strain discrimination. Although draft-level genome sequencing still incurs a substantial cost in relation to some other

typing methods, a reduction of per-sample cost is expected for next-generation sequencing. The analysis of whole genomes would consequently result in a more efficient and effective working methodology, having the potential of replacing multiple individual tests for monitoring pathogen outbreaks and emergence of hyper-virulent strains.7,35

Virulence factors

Virulence factors, implicated in conferring STEC an ability to cause disease in humans, can be found both on the chromosome and plasmids (Table 2).3 The genes coding for Stx, stxl and stx2, are considered to be the primary and defining virulence factor of STEC.4,36,37 Stxs are AB5-type toxins, which consist of a single A-subunit with enzymatic activity and five identical B-subunits with receptor binding ability. The receptors for Stx, globotriaosylceramide (Gb3) or globotetraosylceramide (Gb4), are found on the cell surface of mammalian cells. Stxs have been divided in two major groups, Stx1 and Stx2, and each group has several subtypes. In particular, the Stx1 group is composed of three subtypes, Stx1a, Stx1c and Stx1d; while the Stx2 group is more heterogeneous and diverse and consists of seven subtypes, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f and Stx2g.

Epidemiological studies suggest that STEC strains that are Stx2-positive may be more virulent than those expressing Stx1,4,36,37 and cytotoxicity assays revealed that these subtypes may be associated with different level of virulence.38-40 Studies have documented that STEC expressing Stx1a subtype can potentially cause HUS, while those harboring Stx1c have been associated with mild disease or asymptomatic carriers4,36,37 On the other hand, Stx1d may potentially cause disease in humans and very limited information about its clinical implications is

available. Moreover, molecular typing of STEC strains have shown a strong correlation between strains expressing Stx2a, Stx2b, Stx2c and Stx2d subtypes and severe illness such as bloody diarrhea and HUS. In contrast, STEC strains that are positive for Stx2e subtype have been linked with either mild disease in humans without complications or with asymptomatic carriers. Other subtypes that have had limited association with pathogenesis in humans are Stx2f and Stx2g.

Additional virulence factors present on pathogenicity islands, include the locus of enterocyte effacement (LEE) and the non-LEE effectors, implicated in host colonization and disease (Table 2).3,41 In particular, the LEE-encoded eae gene is considered as a key virulence factor for the attachment to intestinal epithelial cells. An adhesin, iha, the iron-regulated gene A homolog adhesin, may contribute to the attachment of LEE-positive as well as LEE-negative strains. Moreover, the nle effectors, not encoded by the LEE region, have been implicated in altering the host cell response and have been linked to disease severity in non-O157 STEC.3,42 Other chromosomal and plasmid virulence genes, encoding proteases (espP), cytotoxins (subA), and adhesins (saa), may contribute to STEC pathogenesis by allowing bacterial attachment and colonization of the human epithelium (Table 2).3,41 The detection of these virulence genes in STEC strains would provide key information for the identification of risk factors that may potentially contribute to the development of human disease.

Antimicrobial resistance

Several published reports have recently documented an increase in antimicrobial resistance in STEC O157:H7 and non-O157:H7 strains, recovered from domestic animal reservoirs that potentially could impact food and environmental sources (Table 3).19,43—49 In agricultural regions, the inappropriate usages of antibiotics for treating either human or plant diseases and for promoting food-animal growth are proposed to contribute to the continued increase in antimicrobial resistance as well as to the emergence of multidrug resistance profiles.50,51 Moreover, the use of antimicrobials to treat STEC infections is highly controversial since these agents can induce Stx production and thus promoting the onset of HUS in humans.52 For example, sub-inhibitory doses of sulfonamides, quinolones and fluoroquinolones which target DNA synthesis have resulted in an increased production of Stx.53 However, other studies have suggested the early administration in the course of a STEC infection of certain classes of antimicrobials, belonging to macrolides, tetracyclines, fosfomycins, aminoglycosides, cephems and ansamycins. These types of antimicrobials classes target the cell wall, transcription or translation and fail to induce toxin production in STEC.53

In the past year, research publications have highlighted the emergence of novel antimicrobial resistance in zoonotic E. coli, recovered from domestic animal hosts as well as from food of animal origin (Table 3).54 This new resistance is against colistin (polymyxin E), a cationic polypeptide antibiotic that traditionally has been approved for use in food-producing animals due to the low resistance rates

reported.54 The resistance mechanism against colistin is mediated by either the mcr-1 and mcr-2 genes, both transferable on conjugative plasmids.55,56 Following the initial report on plasmid-encoded colistin resistance in pigs,55 a series of published articles have documented transferable resistance against colistin in E. coli isolates from food, livestock, water, humans, and wildlife in multiple countries and five continents.54 Moreover, the identification of plasmid-mediated colistin resistance in STEC pig isolates, which are also resistant to extended-spectrum b-lactamase, has presented new challenges to the veterinary and public health sectors.43 The findings on transferable colistin indicate the urgent need to monitor the use of antimicrobials in animal food production in order to limit the unwanted dissemination of multidrug resistant E. coli.

Conclusions

STEC O157 and non-O157 represents a serious threat to public health worldwide. The potential for large-scale outbreaks and widespread prevalence in animal sources have necessitated the development and evaluation of rapid, sensitive, and specific methods for detection and surveillance for this pathogen. This review summarized the current technology trends for detection and isolation of STEC strains, including culture, isolation, phenotypic, and genotypic characterization methods.

Conflicts of interest

The authors declare no conflicts of interest with the subject matter or materials discussed in the manuscript.

Acknowledgements

This work was supported in part by the United States Department of Agriculture-Agricultural Research Service CRIS project number 2030-42000-051-00D.

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