A Trilocus Sequence Typing Scheme for Hospital Epidemiology and Subspecies Differentiation of an Important Nosocomial Pathogen,Enterococcus faecalis

In this study, we present a trilocus sequence typing (TLST) scheme based on intragenic regions of two antigenic genes, ace and salA (encoding a collagen/laminin adhesin and a cell wall-associated antigen, respectively), and a gene associated with antibiotic resistance, lsa (encoding a putative ABC transporter), for subspecies differentiation of Enterococcus faecalis. Each of the alleles was analyzed using 50 E. faecalis isolates representing 42 diverse multilocus sequence types (ST^M; based on seven housekeeping genes) and four groups of clonally linked (by pulsed-field gel electrophoresis PFGE]) isolates. The allelic profiles and/or concatenated sequences of the three genes agreed with multilocus sequence typing (MLST) results for typing of 49 of the 50 isolates; in addition to the one exception, two isolates were found to have identical TLST types but were single-locus variants (differing by a single nucleotide) by MLST and were therefore also classified as clonally related by MLST. TLST was also comparable to PFGE for establishing short-term epidemiological relationships, typing all isolates classified as clonally related by PFGE with the same type. TLST was then applied to representative isolates (of each PFGE subtype and isolation year) of a collection of 48 hospital isolates and demonstrated the same relationships between isolates of an outbreak strain as those found by MLST and PFGE. In conclusion, the TLST scheme described here was shown to be successful for investigating short-term epidemiology in a hospital setting and may provide an alternative to MLST for discriminating isolates.Enterococci are commensal members of the gastrointestinal tract flora of humans and animals. Within the last 2 decades, enterococci have emerged as the second to third most frequent cause of nosocomial infections, including endocarditis and bloodstream, urinary tract, and wound infections, among others (8, 15, 19, 24, 39). These organisms are also known to have the ability to acquire and transfer antibiotic resistance genes and virulence-associated genes (37). Although there are more than 30 species of the genus Enterococcus, two species, Enterococcus faecalis and Enterococcus faecium, account for a vast majority of enterococcal clinical and nosocomial infections (15, 21, 35). In the past, several molecular typing studies have shown that specific lineages of pathogenic bacteria arise periodically, proliferate, and spread in the presence of selective pressures (34). Therefore, accurate typing of enterococcal strains is crucial for the identification of particular clones capable of causing infections and with the ability to spread in the hospital environment.A number of phenotypic and genotypic typing methods have been applied to the subspecies differentiation of enterococcal isolates. Phenotypic methods which have been used in the past include serotyping (17, 22, 26) and multilocus enzyme electrophoresis (50). Genotypic methods include, among others (3, 52, 53), ribotyping (14, 38), repetitive sequence-based PCR (25, 35), multilocus variable-number tandem-repeat analysis (49, 54), pulsed-field gel electrophoresis (PFGE) (10, 12, 49), and multilocus sequence typing (MLST) (10, 26, 31, 41). Among these methods, PFGE, based on chromosomal restriction endonuclease digestion patterns, is widely used for the study of hospital outbreaks and is considered by many to be the “gold standard” molecular typing technique (48). However, this methodology has several limitations due to the facts that it is labor-intensive and the results have poor interlaboratory transportability. This technique is also unsuitable for long-term epidemiology and population studies due to changes in restriction sites, genomic rearrangements, and/or acquisition of DNA by a clonal lineage, which may markedly change the restriction pattern (41). A more appropriate typing technique for long-term epidemiology, which is currently also widely used for subspecies differentiation, is MLST. MLST, based on the allelic variations in sequences of multiple loci, unambiguously types strains (23) and offers an advantage over other techniques used for typing, such as PFGE, since the data are objective and easily stored, compared, and shared via the Internet.Two different MLST schemes have been used successfully for differentiation of E. faecalis strains (31, 41). The first scheme, which assessed three antigenic genes and one housekeeping gene, found that the allelic profile of two antigenic genes (ace and salA) was sufficient to discriminate the 22 E. faecalis isolates included therein (31). The second MLST scheme, based on the allelic profiles of seven housekeeping genes, was used to type 110 isolates and provided insight into the population structure as well as long-term epidemiological relationships of E. faecalis strains (41). However, typing studies on other organisms, such as Salmonella enterica serovar Typhimurium and Staphylococcus aureus, have suggested that MLST based on housekeeping genes may not provide enough discriminatory power to study hospital outbreaks or to accurately determine short-term genetic relationships, which can be crucial for hospital epidemiology and infection control purposes (9, 13, 27).Our hypothesis for this work was that a sequence-based methodology applied to genes encoding antigenic or cell surface proteins (rather than housekeeping genes) may potentially be more useful to establish short-term epidemiologic relationships in E. faecalis, since these genes would be more susceptible to evolutionary selective pressures and potentially could identify and discriminate isolates from hospital outbreaks, similar to PFGE.In the present work, the trilocus sequence types (ST^T; sequence type based on three genes) of 50 isolates were compared to their multilocus sequence types (ST^M; sequence type based on seven housekeeping genes). To determine the applicability of trilocus sequence typing (TLST) for a clinical setting, the scheme was also used to type sets of predetermined (by PFGE) clones and was then applied to a collection of hospital isolates from Bogota, Colombia, recently reported by Arias et al. to belong to an ST-2 clonal lineage (1).(Part of this work was presented at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007.)