Multilocus Sequence Typing Reveals a Lack of Diversity among Escherichia coli O157:H7 Isolates That Are Distinct by Pulsed-Field Gel Electrophoresis

Abstract

Escherichia coli O157:H7 is a major cause of foodborne illness in the United States. Pulsed-field gel electrophoresis (PFGE) is the molecular epidemiologic method mostly commonly used to identify food-borne outbreaks. Although PFGE is a powerful epidemiologic tool, it has disadvantages that make a DNA sequencebased approach potentially attractive. Multilocus sequence typing (MLST) analyzes the internal fragments of housekeeping genes to establish genetic relatedness between isolates. We sequenced selected portions of seven housekeeping genes and two membrane protein genes (ompA and espA) of 77 isolates that were diverse by PFGE to determine whether there was sufficient sequence variation to be useful as an epidemiologic tool. There was no DNA sequence diversity in the sequenced portions of the seven housekeeping genes and espA. For ompA, all but five isolates had sequence identical to that of the reference strains. E. coli O157:H7 has a striking lack of genetic diversity in the genes we explored, even among isolates that are clearly distinct by PFGE. Other approaches to identify improved molecular subtyping methods for E. coli 0157:H7 are needed.Escherichia coli O157:H7 was first recognized in 1982 in association with a food-borne outbreak and is now recognized as an important cause of food-borne illness in the United States, causing an estimated 74,000 infections a year (20,26). This pathogen causes both bloody diarrhea and hemolytic uremic syndrome (HUS), a severe illness characterized by hemolytic anemia and acute renal failure (2). In recent years, there have been numerous large outbreaks of E. coli O157:H7-related bloody diarrhea and HUS (3,4,11). Most E. coli O157:H7 infections are caused by exposure to food or water that has bovine fecal contamination.Pulsed-field gel electrophoresis (PFGE) is currently the most widely utilized molecular subtyping method for detecting outbreaks of E. coli O157:H7. In fact, PFGE has been found to identify outbreaks of E. coli O157:H7 that were not detected by traditional epidemiologic methods (1). However, DNA sequence-based methods offer several important potential advantages over PFGE, including shorter assay times and fully comparable and transferable data between laboratories (5, 16,19,21). Additionally, while PFGE is relatively simple and inexpensive, it is labor intensive, the interpretation of banding patterns can be subjective, and it does not easily handle large sample sets (13).Multilocus sequence typing (MLST) is a DNA sequencebased molecular subtyping method that has been used successfully for other bacteria, such as Neisseria meningitidis, Streptococcus pneumoniae, and Salmonella, for both evolutionary and epidemiologic studies (8,16,22,29). Briefly, MLST examines the alleles of selected housekeeping genes by nucleotide sequencing a 500-to 600-bp segment of the gene. The sequence data are then analyzed to determine the genetic relatedness of the bacterial isolates. In this study, we performed MLST on a set of E. coli O157:H7 isolates that had been characterize...

Vibrio cholerae Strain Typing and Phylogeny Study Based on Simple Sequence Repeats

“…Both methods revealed the same number of alleles; however, the same degree of nucleotide variation was found on shorter segments (115 to 270 bp) of the MNR loci compared to a longer segment (560 to 1,100 bp) for the housekeeping, 16S-23S rRNA intergenic spacer regions or virulence genes (10,13,29,44). The original MLST method is based on sequence variation at housekeeping genes that are conserved and therefore usually present in all strains but that have limited variation (21,59,69). On the other hand, the MNR loci that present high levels of polymorphism have the disadvantage of missing data from a portion of the strains due to nonamplified ("null") alleles (20,75) as a result of sequence mismatch (SNPs) at the priming sites, as well as insertions or deletions and genome rearrangements (32).…”

“…The variation in SSR tracts, generally having several alleles, enables their use for strain typing in several bacterial species (30,38,41,59,80). SSR composed of mononucleotide repeats (MNR) in Escherichia coli were found to be abundant and highly polymorphic but stable at the strain level, making them a valuable tool for bacterial typing and phylogenetics studies (20,30).…”

Insertions, Deletions, and Single-Nucleotide Polymorphisms at Rare Restriction Enzyme Sites Enhance Discriminatory Power of Polymorphic Amplified Typing Sequences, a Novel Strain Typing System for Escherichia coli O157:H7

“…DNA sequence-based typing methods, such as multilocus sequence typing, are potential alternatives for PFGE, as they offer shorter assay times, less subjectivity in the interpretation of results, comparable and transferable data, and ease of automation (25). However, these techniques still rely on expensive instrumentation and software to interpret data (29).…”

“…Most importantly, these typing systems are inefficient in global analysis of the genome, showing a bias toward a select set of sequences or genes that may or may not have sufficient diversity among strains of a pathogen. Also, sequencing errors may influence the results (25).…”

Detection and epidemiology of plasmid-mediated AmpC β-lactamase producing Escherichia coli in two Irish tertiary care hospitals

“…Two isolates (isolates E90 and E91) had the same allelic profiles and therefore the same ST (ST720). These two isolates only displayed 40% similarity by PFGE, highlighting that MLST has a lower discriminating ability compared with PFGE, as reported in previous studies [26][27][28]. The remaining seven isolates had different individual allelic profiles and therefore different STs.…”