Development of an allele-specific PCR for Escherichia coli B2 sub-typing, a rapid and easy to perform substitute of multilocus sequence typing

Clermont, Olivier; Christenson, Julia K.; Daubié, Anne-Sophie; Gordon, David M.; Denamur, Erick

doi:10.1016/j.mimet.2014.03.008

Cited by 74 publications

(53 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We sequenced the rpoS gene in the NILS collection (see Fig. S1 in the supplemental material) to verify that this congruence between species and rpoS phylogeny was not limited to the IAI collection, and we divided the strains into the main phylogroups and D and B2 subgroups by PCR-based approaches (32,(35)(36)(37). Once again, we observed very good congruence between the rpoS gene and phylogroups and subgroups.…”

Section: Resultsmentioning

confidence: 79%

“…IAI and NILS strains were classified according to the seven main phylogroups of the species, and strains belonging to Escherichia clade I were identified as described in references 34 and 35. The clonal group A (CGA) was identified among the D strains as described in reference 36, and the B2 group strains were assigned to the main B2 subgroups (I to X) with MLST data for the IAI collection (32) and allele-specific PCR for the NILS collection (37). O typing was performed with an allele-specific PCR assay (38,39).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

The rpoS Gene Is Predominantly Inactivated during Laboratory Storage and Undergoes Source-Sink Evolution in Escherichia coli Species

et al. 2014

Self Cite

View full text Add to dashboard Cite

The rpoS gene codes for an alternative RNA polymerase sigma factor, which acts as a general regulator of the stress response. Inactivating alleles of rpoS in collections of natural Escherichia coli isolates have been observed at very variable frequencies, from less than 1% to more than 70% of strains. rpoS is easily inactivated in nutrient-deprived environments such as stab storage, which makes it difficult to determine the true frequency of rpoS inactivation in nature. We studied the evolutionary history of rpoS and compared it to the phylogenetic history of bacteria in two collections of 82 human commensal and extraintestinal E. coli strains. These strains were representative of the phylogenetic diversity of the species and differed only by their storage conditions. In both collections, the phylogenetic histories of rpoS and of the strains were congruent, indicating that horizontal gene transfer had not occurred at the rpoS locus, and rpoS was under strong purifying selection, with a ratio of the nonsynonymous mutation rate (Ka) to the synonymous substitution rate (Ks) substantially smaller than 1. Stab storage was associated with a high frequency of inactivating alleles, whereas almost no amino acid sequence variation was observed in RpoS in the collection studied directly after isolation of the strains from the host. Furthermore, the accumulation of variations in rpoS was typical of source-sink dynamics. In conclusion, rpoS is rarely inactivated in natural E. coli isolates within their mammalian hosts, probably because such strains rapidly become evolutionary dead ends. Our data should encourage bacteriologists to freeze isolates immediately and to avoid the use of stab storage.T he rates of recombination and mutation in a genome are critical issues because the diversity generated by these mechanisms is the substrate for selection. Some regions of bacterial chromosomes are more prone to mutation (1-4) or recombination (5) than other regions, due to physical or physiological constraints. Then, selection can favor diversification, for example, that of cell surface proteins such as beta barrel porins (6), and recombination events, which can restore particular functions, as in the case of the mismatch repair (MR) genes (7,8).There is growing evidence that selection also acts on central regulators to allow adaptation by rewiring of networks. In vitro experimental models of Escherichia coli evolution have shown that adaptation occurs through modifications of global regulatory networks, which are often interconnected (9-13). These networks control DNA superhelicity (12), the stringent response (12), and the general stress response, via RpoS (9, 10, 13). RpoS is an alternative sigma factor of RNA polymerase that regulates more than 10% of E. coli genes and plays a critical role in survival under conditions of exposure to several types of stress, including acid, heat, and oxidative stress (14,15). A particular case of in vitro evolution is the inactivation of crl (which modulates the balance between different sigma f...

show abstract

Section: Resultsmentioning

confidence: 79%

Section: Methodsmentioning

confidence: 99%

The rpoS Gene Is Predominantly Inactivated during Laboratory Storage and Undergoes Source-Sink Evolution in Escherichia coli Species

et al. 2014

Self Cite

View full text Add to dashboard Cite

show abstract

“…Isolates that qualified as ExPEC, plus a 2:1 randomly selected comparison set of non-ExPEC isolates, were tested by established PCR-based assays for E. coli phylogroup (A, B1, B2, C, D, E, and F) (32) (33)(34)(35), and 43 additional virulence-associated genes or variants thereof (27). ST131 isolates were additionally assessed for membership in the ST131-H30 subclone and for the (ST131-associated) O25b and O16 rfb variants using established PCR-based assays (4,24).…”

Section: Methodsmentioning

confidence: 99%

Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli, Including Sequence Type 131 (ST131), from Retail Chicken Breasts in the United States in 2013

Johnson

Porter

Johnston

et al. 2017

Appl Environ Microbiol

View full text Add to dashboard Cite

Chicken meat products are hypothesized to be vehicles for transmitting antimicrobial-resistant and extraintestinal pathogenic Escherichia coli (ExPEC) to consumers. To reassess this hypothesis in the current era of heightened concerns about antimicrobial use in food animals, we analyzed 175 chicken-source E. coli isolates from a 2013 Consumer Reports national survey. Isolates were screened by PCR for ExPEC-defining virulence genes. The 25 ExPEC isolates (12% of 175) and a 2:1 randomly selected set of 50 non-ExPEC isolates were assessed for their phylogenetic/ clonal backgrounds and virulence genotypes for comparison with their resistance profiles and the claims on the retail packaging label ("organic," "no antibiotics," and "natural"). Compared with the findings for non-ExPEC isolates, the group of ExPEC isolates had a higher prevalence of phylogroup B2 isolates (44% versus 4%; P Ͻ 0.001) and a lower prevalence of phylogroup A isolates (4% versus 30%; P ϭ 0.001), a higher prevalence of multiple individual virulence genes, higher virulence scores (median, 11 [range, 4 to 16] versus 8 [range, 1 to 14]; P ϭ 0.001), and higher resistance scores (median, 4 [range, 0 to 8] versus 3 [range, 0 to 10]; P Ͻ 0.001). All five isolates of sequence type 131 (ST131) were ExPEC (P ϭ 0.003), were as extensively resistant as the other isolates tested, and had higher virulence scores than the other isolates tested (median, 12 [range, 11 to 13] versus 8 [range, 1 to 16]; P ϭ 0.005). Organic labeling predicted lower resistance scores (median, 2 [range, 0 to 3] versus 4 [range, 0 to 10]; P ϭ 0.008) but no difference in ExPEC status or virulence scores. These findings document a persisting reservoir of extensively antimicrobial-resistant ExPEC isolates, including isolates from ST131, in retail chicken products in the United States, suggesting a potential public health threat.IMPORTANCE We found that among Escherichia coli isolates from retail chicken meat products purchased across the United States in 2013 (many of these isolates being extensively antibiotic resistant), a minority had genetic profiles suggesting an ability to cause extraintestinal infections in humans, such as urinary tract infection, implying a risk of foodborne disease. Although isolates from products labeled "organic" were less extensively antibiotic resistant than other isolates, they did not appear to be less virulent. These findings suggest that retail chicken products in the United States, even if they are labeled "organic," pose a potential health threat to consumers because they are contaminated with extensively antibiotic-resistant and, presumably, virulent E. coli isolates.

show abstract

“…The new E. coli STc648-specific assay can be used in conjunction with published assays that detect ST131 and its subsets (40-42), eight other major clonal subsets within phylogroup B2 (43), and three important phylogroup D-derived clonal subsets (ST69, the O15:K52:H1-ST31/ST393 complex, and ST405) (44)(45)(46), for an extensive clonal analysis of extraintestinal E. coli isolates. Because several of these lineages are typically multidrug-resistant (4), such clonal typing may help to identify reservoirs of resistance, track epidemiological trends, and guide empirical antimicrobial therapy selection (8).…”

Section: Discussionmentioning

confidence: 99%

“…STc648 was defined operationally as including ST648 and its single-and double-locus variants. To develop the STc648-specific assay, we used an approach similar to that we used for developing other STc-specific PCR-based assays (40,41,(43)(44)(45). First, we assessed the STc648-defining alleles at each of the seven Achtman MLST loci for specificity (or quasi-specificity) to STc648.…”

Section: Methodsmentioning

confidence: 99%

Rapid and Specific Detection of the Escherichia coli Sequence Type 648 Complex within Phylogroup F

2017

Self Cite

View full text Add to dashboard Cite

The Escherichia coli sequence type 648 complex (STc648) is an emerging lineage within phylogroup F-formerly included within phylogroup D-that is associated with multidrug resistance. Here, we designed and validated a novel multiplex PCR-based assay for STc648 that took advantage of (i) four distinctive single-nucleotide polymorphisms in icd allele 96 and gyrB allele 87, two of the multilocus sequence typing alleles that define ST648; and (ii) the typical absence within STc648 of uidA, an E. coli-specific gene encoding ␤-glucuronidase. Within a diverse 212-strain validation set that included 109 STs other than STc648, from phylogroups A, B1, B2, C, D, E, and F, the assay exhibited 100% sensitivity (95% confidence interval [CI], 82% to 100%) and specificity (95% CI, 98% to 100%). It functioned similarly well in two distant laboratories that used boiled lysates or DNAzol-purified DNA as the template DNA. Thus, this novel multiplex PCR-based assay should enable any laboratory equipped for diagnostic PCR to rapidly, accurately, and economically screen E. coli isolates for membership in STc648.KEYWORDS Escherichia coli, antimicrobial resistance, diagnostics, molecular epidemiology, polymerase chain reaction, sequence type, strain typing E scherichia coli, an important cause of extraintestinal infections in humans and animals (1), is highly clonal. Each of its seven recognized phylogenetic groups (phylogroups) comprises numerous individual sequence types (STs), as defined by multilocus sequence typing (MLST) (2). Among thousands of distinct E. coli STs, a dozen or so ST complexes (i.e., groups of closely related STs) account for most human extraintestinal E. coli infections, and so are regarded as extraintestinal pathogenic E. coli (ExPEC) (3, 4); a similarly small proportion account for most antimicrobial-resistant E. coli infections (4-7). Thus, an E. coli isolate's ST can be highly informative regarding its likely pathogenic and resistance capabilities (8, 9).Phylogroup F is related closely to phylogroup B2, the origin of most human clinical E. coli isolates, and phylogroup D, the origin of most non-B2 ExPEC strains (2, 10). Prior to its recognition as a distinct phylogroup, its members were usually classified under group D, including by a PCR-based phylotyping assay that delineates only four major E. coli phylogroups (11). An updated version of that assay differentiates phylogroup F from phylogroup D (2).Within phylogroup F, the sequence type 648 complex (STc648) is reported increasingly as an emerging resistance-associated lineage (4). In multiple studies of resistant E. coli from diverse sources and locales, STc648 has been the first, second, or third most prevalent STc, accounting for up to 28% of isolates (12)(13)(14)(15)(16)(17)(18)(19). The reported resistance phenotypes for different STc648 strains include fluoroquinolones, extended-spectrum cephalosporins (CTX-M-type enzymes and CMY-2), carbapenems (OXA-48 and NDM and KPC variants), fosfomycin (fosA3), and colistin (mcr-1) (13, 14, 20-30). STc648 is...

show abstract

Development of an allele-specific PCR for Escherichia coli B2 sub-typing, a rapid and easy to perform substitute of multilocus sequence typing

Cited by 74 publications

References 29 publications

The rpoS Gene Is Predominantly Inactivated during Laboratory Storage and Undergoes Source-Sink Evolution in Escherichia coli Species

The rpoS Gene Is Predominantly Inactivated during Laboratory Storage and Undergoes Source-Sink Evolution in Escherichia coli Species

Extraintestinal Pathogenic and Antimicrobial-Resistant Escherichia coli, Including Sequence Type 131 (ST131), from Retail Chicken Breasts in the United States in 2013

Rapid and Specific Detection of the Escherichia coli Sequence Type 648 Complex within Phylogroup F

Contact Info

Product

Resources

About