Susceptibility of vancomycin-resistant and -sensitive Enterococcus faecium obtained from Danish hospitals to benzalkonium chloride, chlorhexidine and hydrogen peroxide biocides

Efflux pump genes and proteins are present in both antibiotic-susceptible and antibiotic-resistant bacteria. Pumps may be specific for one substrate or may transport a range of structurally dissimilar compounds (including antibiotics of multiple classes); such pumps can be associated with multiple drug (antibiotic) resistance (MDR). However, the clinical relevance of efflux-mediated resistance is species, drug, and infection dependent. This review focuses on chromosomally encoded pumps in bacteria that cause infections in humans. Recent structural data provide valuable insights into the mechanisms of drug transport. MDR efflux pumps contribute to antibiotic resistance in bacteria in several ways: (i) inherent resistance to an entire class of agents, (ii) inherent resistance to specific agents, and (iii) resistance conferred by overexpression of an efflux pump. Enhanced efflux can be mediated by mutations in (i) the local repressor gene, (ii) a global regulatory gene, (iii) the promoter region of the transporter gene, or (iv) insertion elements upstream of the transporter gene. Some data suggest that resistance nodulation division systems are important in pathogenicity and/or survival in a particular ecological niche. Inhibitors of various efflux pump systems have been described; typically these are plant alkaloids, but as yet no product has been marketed.

Introduction In the past two decades, Enterococcus faecium has become recognized as an important nosocomial pathogen. Up to the 1980s, the large majority of enterococcal hospital-associated infections (HAI) were caused by Enterococcus faecalis, but since the beginning of the 1990s, the proportion of HAI caused by E. faecium has increased and has now almost reached parity with that of E. faecalis (1). One proposed reason for this changing epidemiology is that, in comparison with E. faecalis, E. faecium shows relatively high rates of resistance against important antibiotics, including ampicillin and vancomycin. In addition, studies of the population biology of E. faecium using multilocus sequence typing (MLST) data have revealed the existence of a distinct genetic subpopulation associated with nosocomial epidemics. This subpopulation has been designated lineage C1 (2) and was later renamed clonal complex 17 (CC17) on the basis of eBURST (3) analysis of MLST data (4, 5). CC17 has been recognized as a successful hospital-associated E. faecium (HA E. faecium) clonal complex, exhibiting high-level vancomycin, ampicillin, and quinolone resistance, although in most European countries CC17 remained primarily vancomycin susceptible (4, 6, 7, 8, 9, 10, 11, 12, 13). However, in addition to this distinct resistance profile, genome-wide analyses have shown that HA E. faecium strains have a genetic repertoire distinct from that of E. faecium strains that asymptomatically colonize the gastrointestinal (GI) tract of humans and animals in the community (14, 15). This distinct genetic repertoire includes cell surface proteins, of which the enterococcal surface protein, Esp, is a known virulence determinant (8, 10, 16, 17, 18, 19, 20, 21, 22, 23); genomic islands tentatively encoding novel metabolic pathways (24); and insertion sequence elements (14). It is now considered that these determinants may be adaptive elements that have improved the relative fitness of this HA E. faecium subpopulation in the hospital environment (5, 9, 25, 26). Despite the clear importance of CC17 as the main genetic subpopulation, including hospital isolates, its precise phylogenetic status remains uncertain. Analyses of the population structure of E. faecium indicate that this species undergoes a high rate of homologous recombination (4, 27). High recombination rates can lead to error in phylogenetic analyses. This is especially true if only a small portion of the genome is interrogated. In the case of the eBURST approach used to define CC17, a consequence of large amounts of recombination is the spurious grouping of diverse and distinct lineages into a single clonal complex. It has previously been suggested (28) that phylogenetic reconstructions of E. faecium are vulnerable to such errors. Analyses using alternative methods to eBURST suggest this may be the case, with the constituent lineages of CC17 (sequence types [STs] 17, 18, 78, and 80) representing distinct genetic lineages of which the relationships between cannot be confidently assigned (5, 26, 27). Together with the observation that whole genome sequences of two CC17 isolates (E1162 [ST17] and U0317 [ST78]) differ substantially in gene content (15), this strongly indicates that HA E. faecium may not have evolved from a single founder (i.e., ST17) and that, consequently, CC17 as presently identified may not exist but is an artifact of the assumptions embedded within the eBURST algorithm. An alternative approach to eBURST is an analysis of genetic population structure, with the power to combine the identification of deep branching lineages and recombination between them. This can be done using Bayesian Analysis of Population Structure (BAPS) software (29, 30, 31). Unlike approaches such as eBURST (3), BAPS does not attempt to retrieve phylogenetic information or implement a phylogenetic model of clustering but rather uses a statistical genetic model to partition molecular variation based on both clonal ancestry and recombination patterns as identified from DNA sequence data. This approach has recently been used to probe genetic population structure in Streptococcus pneumoniae (32), Escherichia coli (33), Campylobacter coli, Campylobacter jejuni (34, 35), and Neisseria spp. (36) and has been shown to be able to detect structure even in highly recombinogenic populations. Here, we used BAPS to identify groups of related E. faecium strains and show a significant association of hospital and farm animal isolates to different BAPS groups. We suggest that hospital-associated lineages contained in different BAPS groups have, however, acquired similar adaptive elements. RESULTS BAPS-based genetic structure in the E. faecium population. The data set used for our analysis consisted of 519 distinct STs, of which 491 were found among 1,720 E. faecium isolates and the remaining 28 STs in 29 E. faecalis isolates. The eBURST analysis based on the 491 E. faecium STs yielded 19 eBURST groups and 101 singletons (STs not part of an eBURST group or clonal complex) (Fig. 1). The largest eBURST group included 329 (67%) of the STs and 1,459 (85%) of the isolates, indicating that eBURST might be unreliable in estimating relatedness in this large straggly E. faecium group. FIG 1 eBURST-based population snapshot of E. faecium based on 519 STs representing 1,749 isolates contained in the online E. faecium MLST database (http://efaecium.mlst.net). STs belonging to lineages 17, 18, and 78 are color coded in blue, red, and yellow, respectively. To gain an alternative perspective on E. faecium population structure, we used a Bayesian population genetic model to identify clusters characterized by different allele frequencies based on multilocus DNA sequences (31). The 519 STs were partitioned into 7 groups, of which groups 1 to 6 included the E. faecium isolates, while BAPS group 7 was entirely composed of E. faecalis isolates (Table 1). BAPS groups 2 and 3 represented the majority of the sample, containing 227 (44%) and 190 (40%) of the STs and 829 (47%) and 784 (45%) of the isolates, respectively (Table 1). It is known that in Bayesian model-based analysis, fine distinctions between closely related clusters can be obscured by the presence of relatively distant clusters in the data, which produce a large amount of signal and consequently render smaller signals of difference insignificant (see, e.g., the discussion in the work of Marttinen et al. [37]). As a result, BAPS groups 2 and 3 were each individually analyzed using BAPS. This nested analysis strategy was implemented to provide greater resolution of population structure in these groups. The results showed four subgroups of BAPS group 2 (BAPS 2-1 to 2-4) and five subgroups of BAPS group 3 (BAPS 3-1 to 3-5) (Table 1). The nested analysis thus finally subdivided the E. faecium population into 13 distinct subpopulations and one E. faecalis BAPS group 7. Given the smaller sizes of the subgroups emerging in the second stage of clustering compared to the initial analysis, it is not feasible to split these subgroups even further by successively repeating the clustering procedure, as there will not be enough variation in the data to reliably infer the underlying groups. TABLE 1 Distribution of STs and isolates among BAPS subpopulations BAPSgroup BAPSsubgroup No.of STs No. of E. faecium isolates from: No.of E. faecalis isolates Totalno. of isolates Hospitalpatients Nonhospitalpersons Pets Pigs Poultry Calves Other a Unknown 1 53 36 12 0 10 0 0 11 14 0 83 2 1 148 403 18 71 34 97 0 20 30 0 673 2 1 1 0 0 0 0 0 0 0 0 1 3 63 74 9 7 7 3 17 6 5 0 128 4 15 15 0 1 1 3 0 5 2 0 27 3 1 44 53 6 7 8 1 0 8 3 0 86 2 17 13 9 4 14 1 0 1 6 0 48 3 104 547 10 8 6 12 3 8 10 0 604 4 4 2 4 0 1 0 0 0 0 0 7 5 21 21 1 9 0 4 0 2 2 0 39 4 9 6 1 0 0 0 0 1 1 0 9 5 9 5 0 0 2 2 0 0 1 0 10 6 3 4 0 0 0 0 0 0 1 0 5 7 28 0 0 0 0 0 0 0 0 29 29 Total 519 1,180 70 107 83 123 20 62 75 29 1,749 a Includes animal food products (n = 29), other animal isolates, or isolates from nonspecified animals (n = 8) and environmental isolates (n = 25). Distribution of E. faecium isolates among BAPS (sub)groups. Two BAPS groups (2-1, 3-3) contained the majority (80%) of isolates from hospitalized patients (Table 1). However, BAPS 2-1 also contained a large number of animal-related isolates, in contrast with BAPS 3-3. To assess significance of associations between BAPS groups and isolate sources, odds ratios (ORs) were calculated. This revealed a significant association of hospital and farm animal isolates to different BAPS groups. Strikingly, BAPS group 3-3 was positively associated with isolates retrieved from hospitalized patients, whereas hospital isolates were negatively associated with BAPS groups 1, 2-1, 2-3, 3-2, and 3-4. In turn, isolates from farm animals were significantly associated with BAPS groups 2-1, 2-3, 2-4, 3-2, and 5 and negatively associated with BAPS group 3-3 (see Table 2). To examine whether sampling bias could influence results by inflating the numbers of individual STs, ORs were calculated based on a single example of each ST. The results confirmed positive association of BAPS 3-3 with hospitalized patients and BAPS 2-1 and 2-4 with farm animals and negative association of BAPS 2-1 and 3-2 with hospitalized patients and BAPS 3-3 with farm animals (see Table S2 in the supplemental material). TABLE 2 Associations between E. faecium isolates from hospitalized patients and farm animals with BAPS groups and lineages Source BAPS group Lineage No. of isolates from source Total no. of isolates a ORs c 95% CI Hospitalized patients 1 36 69 0.41 0.254–0.669 2-1 allSTs 403 643 0.49 0.391–0.605 2-1 78 364 453 1.89 1.45–2.449 2-2 1 1 ND 2-3 74 123 0.57 0.389–0.829 2-4 15 25 0.59 0.261–1.314 3-1 53 83 0.68 0.43–1.082 3-2 13 42 0.17 0.086– 0.325 3-3 allSTs 547 594 7.69 5.567–10.61 3-3 17 329 342 13.44 7.633–23.67 3-3 18 190 196 14.69 6.465–33.341 3-4 2 7 0.16 0.03–0.808 3-5 21 37 0.51 0.263–0.983 4 6 8 1.18 0.238–5.883 5 5 9 0.49 0.131–1.834 6 4 4 ND Farm animals b 1 14 69 1.54 0.743–2.477 2-1 allSTs 144 643 2.14 1.64–2.795 2-1 78 4 453 0.03 0.012–0.087 2-2 0 1 ND 2-3 30 123 1.79 1.156–2.756 2-4 9 25 3.03 1.322–6.921 3-1 13 83 0.98 0.531–1.789 3-2 16 42 3.38 1.786–6.39 3-3 allSTs 26 594 0.16 0.103–0.239 3-3 17 2 342 0.02 0.006– 0.095 3-3 18 2 196 0.05 0.012–0.19 3-4 1 7 0.88 0.105–7.301 3-5 6 37 1.02 0.42–2.463 4 0 8 ND 5 4 9 4.25 1.135–15.945 6 0 4 ND a E. faecium isolates in the source categories of hospitalized patients, farm animals, and other sources. Isolates from unknown sources were not included. In total, 1,645 isolates were included in the analysis. b Pigs, poultry, veal calves, meat, and milk products. c ORs indicate significance of association between E. faecium source categories and BAPS (sub)group and lineage. ND, not done. STs 17, 18, and 78 are important nodes in the previously described CC17, a globally distributed clonal complex identified by eBURST analysis (Fig. 1). To correct for erroneous linkage introduced by eBURST in organisms like E. faecium, which have a high recombination-to-mutation ratio (28), we have divided CC17 into lineages arising from each of the subfounder STs (shown in Fig. 1), namely, STs 17, 18, and 78. Two of these (STs 17 and 18, together with descendant STs) are included in BAPS 3-3, which has the strongest and most significant association with hospital-associated isolates among all these groups. eBURST analysis of STs belonging to the two largest BAPS groups, BAPS 2-1 and BAPS 3-3, revealed coclustering of STs representing the majority of hospital-associated E. faecium isolates (881; 74%) into three major lineages: lineage 78 (364 isolates; 42 STs) in BAPS 2-1 and lineage 17 (328 isolates; 35 STs) and lineage 18 (189 isolates; 26 ST) in BAPS 3-3 (see Fig. S1 and S2 in the supplemental material). ORs confirmed a significant association of lineages 17, 18, and 78 with hospital isolates and a significant negative association with isolates from farm animals and meat products (Table 2; see also Table S2 in the supplemental material). Comparison of BAPS-based grouping and whole-genome phylogenomics. BAPS grouping is based on the concatenated sequences of the seven MLST housekeeping genes, which consist of 3,463 bp of DNA sequence. To examine whether the groupings we find are concordant with those based on a larger fraction of the genome, we constructed a phylogenetic tree based on 299 orthologous proteins representing 64,555 amino acids contained in the 29 E. faecium strains for which a genome sequence is available. The phylogenomics analysis showed agreement between BAPS assignment of STs and their relative position in the phylogenetic tree (Fig. 2). Isolates belonging to BAPS 1 clustered far from BAPS 2 and 3 isolates, and distinct groups of isolates corresponding to the BAPS 2-1 and 3-3 subgroups were observed. This suggests that the groupings identified by BAPS analysis of MLST loci reflect a deep phylogenetic structure that is also apparent in larger samples of loci and hints toward functional differences between the subgroups. The phylogenomic tree suggests one monophyletic group consisting of isolates belonging to the three hospital-adapted lineages 17, 18, and 78. However, despite the increased amounts of data available for the construction of the phylogenomic tree in Fig. 2, it must be treated with caution, because sample size and the sampling frame for Fig. 2 is markedly different from that available for MLST data. A neighbor-joining tree based on concatenated alignments of MLST gene sequences contained in the two largest BAPS (sub)groups, BAPS 2 and BAPS 3, clearly indicate phylogenetic diversity between BAPS 2 (lineage 78) and BAPS 3 (lineages 17 and 18) (see Fig. S3 in the supplemental material). FIG 2 Minimum evolution tree based on the concatenated alignments of 299 orthologous proteins conserved in draft genome sequences of 29 E. faecium isolates. Bootstrap values are indicated and are based on 1,000 permutations. Strain codes are indicated as well as ST, BAPS group, and lineage based on their ST. Accession numbers: E. faecium 1231408, GenBank NZ_ACBB00000000; E. faecium 1231501, GenBank NZ_ACAY00000000; E. faecium TX0133A, GenBank NZ_AECH00000000; E. faecium TX0082, GenBank NZ_AEBU00000000; E. faecium C68, GenBank NZ_ACJQ00000000; E. faecium 1231410, GenBank NZ_ACBA00000000; E. faecium 1230933, GenBank NZ_ACAS00000000; E. faecium E4453, GenBank AEDZ00000000; E. faecium 1231502, GenBank NZ_ACAX00000000; E. faecium E4452, GenBank AEOU00000000; E. faecium D344, GenBank NZ_ACZZ00000000; E. faecium TC6, GenBank NZ_ACOB00000000; E. faecium Com15, GenBank NZ_ACBD00000000; E. faecium 1141733, GenBank NZ_ACAZ00000000; E. faecium PC4.1, GenBank NZ_ADMM00000000; E. faecium Com12, GenBank NZ_ACBC00000000; E. faecium TX1330, GenBank NZ_ACHL00000000; E. faecium E980, GenBank ABQA00000000; E. faecium E1039, GenBank ACOS00000000; E. faecium E1071, GenBank ABQI00000000; E. faecium E1162, GenBank ABQJ00000000; E. faecium E1636, GenBank ABRY00000000; E. faecium E1679, GenBank ABSC00000000; E. faecium U0317, GenBank ABSW00000000; E. faecium DO, GenBank NZ_ACIY00000000.1; E. faecium TX133a01, GenBank NZ_AECJ00000000.1; E. faecium TX133a04, GenBank NZ_AEBC00000000.1; E. faecium TX133B, GenBank NZ_AECI00000000.1; E. faecium TX133C, GenBank NZ_AEBG00000000.1. Strains 1231408, PC4.1, and Com15 lack a BAPS (sub)group designation because STs extracted from the genome sequences of these strains were not assigned yet at the time the BAPS analysis was performed. To improve resolution of the upper part of the tree, the top 22 non-BAPS 1 strains were separately clustered using the minimum evolution method. Evolution of the hospital-associated E. faecium population. The fact that animals, specifically poultry and pet animals, are significantly associated with BAPS 2-1 and hospital-associated isolates with BAPS 3-3 suggests a distinction between these two habitats. However, approximately a third (31%) of isolates from hospitalized patients, including the major hospital lineage 78, cocluster with isolates from animal sources in BAPS 2-1 (Table 1). We interpret this as evidence that this subset of hospital-associated isolates has emerged separately from hospital isolates in BAPS 3-3 and has a distinct evolutionary history. Further examination of the constituent lineages of CC17 (i.e., lineages 17 and 18 in BAPS groups 3-3 and lineage 78 in 2-1) suggests that other important clinical features, such as ampicillin resistance and the presence of the esp virulence factor, which is implicated in biofilm formation, urinary tract infections, and endocarditis (17, 22, 23), are common. The esp gene is recorded as present in 73% and 72% of hospital isolates in lineage 17 (n = 248) and 78 (n = 363) isolates, respectively. In contrast, only 31% of lineage 18 isolates (n = 154) are recorded as having this gene present, which compares with 18% in the remainder of the population for which esp presence or absence was recorded (n = 404 isolates). The other feature that is present at increased frequency in all three hospital-associated lineages is ampicillin resistance, which is practically ubiquitous among lineage 17, 18, and 78 hospital isolates (more than 98% of isolates belonging to one of these three lineages are resistant) but, in contrast, is less frequent among the rest of the data set (35%). In a previous publication, using a mixed whole-genome array, we identified additional genes that were enriched among hospital isolates (14). Five genes that ranked highest as genes predictive for hospital isolates in a character evolution analysis in this previous study were also enriched among lineage 17, 18, and 78 hospital isolates (63 to 100%), while being relatively rare among the remainder of the E. faecium population (10 to 30%) (see Table S3 in the supplemental material). Also, SgrA, a nidogen-binding LPXTG surface adhesion implicated in biofilm formation, and EcbA, a collagen binding MSCRAMM (21), are significantly enriched among isolates belonging to the three hospital lineages as well as a genomic island tentatively encoding a metabolic pathway involved in carbohydrate transport and metabolism (24) (see Table S3). We propose that the enrichment of esp in two of the hospital-adapted lineages, which include BAPS groups 3-3 and 2-1, is a result of separate acquisition leading to selection in hospitals and that the same applies to ampicillin resistance and the seven additional genes and the genomic island. Admixture analysis and gene flow networks in E. faecium and between E. faecium and E. faecalis. The BAPS program was used to identify cases of admixture in which genotypes (STs) contain sequences characteristic of more than one subpopulation. For BAPS groups 2 and 3, the vast majority of genotypes have sequence signatures of only one BAPS group, which is indicative of fairly restricted recombination between these groups (Fig. 3A and B; see also Fig. S4 in the supplemental material). Nevertheless, the gene flow diagram shows admixture between BAPS 2 and BAPS 3, with almost 5% of the sequences in BAPS 3 having characteristics of BAPS 2, while for genotypes in BAPS 2, only 0.25% of the sequences have characteristics of BAPS 3. In BAPS groups 1, 4, and 6, at least 94% of the sequences are characteristic of that group, pointing toward restricted recombination also in these BAPS groups (significance of admixture was determined for each ST using the threshold of P values of 0.45 means that at least 45% of the concatenated MLST gene sequences of an ST has sequence characteristics of more than one BAPS group. Each column represents the fraction of STs that belong to a particular category of admixture proportions. Colors indicate BAPS groups to which STs belong. Download Figure S4, TIF file, 0.3 MB. Figure S4, TIF file, 0.3 MB Table S1 Metadata of isolates included in this study. Table S1, PDF file, 0.1 MB. Table S2 Associations between E. faecium STs from hospitalized patients and farm animals and BAPS groups and lineages. Table S2, PDF file, 0.1 MB. Table S3 Distribution of genes enriched among the three hospital lineages. Table S3, PDF file, 0.1 MB. Table S4 Distribution of STs with significant admixture among BAPS groups and source of isolation. Table S4, PDF file, 0.1 MB.