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J Clin Microbiol. Apr 2009; 47(4): 896–901.
Published online Feb 4, 2009. doi:  10.1128/JCM.02283-08
PMCID: PMC2668297

Distribution of Genes Encoding MSCRAMMs and Pili in Clinical and Natural Populations of Enterococcus faecium[down-pointing small open triangle]

Abstract

Enterococcus faecium has recently emerged as an important cause of nosocomial infections. We previously identified 15 predicted surface proteins with characteristics of MSCRAMMs and/or pili and demonstrated that their genes were frequently present in 30 clinical E. faecium isolates studied; one of these, acm, has been studied in further detail. To determine the prevalence of the other 14 genes among various E. faecium populations, we have now assessed 433 E. faecium isolates, including 264 isolates from human clinical infections, 69 isolates from stools of hospitalized patients, 70 isolates from stools of community volunteers, and 30 isolates from animal-related sources. A variable distribution of the 14 genes was detected, with their presence ranging from 51% to 98% of isolates. While 81% of clinical isolates carried 13 or 14 of the 14 genes tested, none of the community group isolates and only 13% of animal isolates carried 13 or 14 genes. The presence of these genes was most frequent in endocarditis isolates, with 11 genes present in all isolates, followed by isolates from other clinical sources. The number of genes significantly associated with clinical versus fecal or animal origin (P = 0.04 to <0.0001) varied from 10 to 13, depending on whether comparisons were made against individual clinical subgroups (endocarditis, blood, and other clinical isolates) or against all clinical isolates combined as one group. The strong association of these genes with clinical isolates raises the possibility that their preservation/acquisition has favored the adaptation of E. faecium to nosocomial environments and/or patients.

Enterococci, commensal members of the intestinal flora in humans and animals, have emerged over the last 3 decades as important hospital-associated opportunistic pathogens causing a variety of infections, including urinary tract infections, surgical site infections, bacteremia, and infective endocarditis (20, 21). Several species of enterococci have been recognized, but more than 90% of infections are caused by only two species, Enterococcus faecalis and Enterococcus faecium. Until the 1990s, infections due to E. faecalis were reported to outnumber those due to E. faecium by 9 to 1, but more recently, the incidence of E. faecium has increased dramatically, and it has been found as the causative organism in 20 to 36% of enterococcal infections in some U.S. hospitals (8, 14, 25, 33). Similarly, hospital-associated E. faecium infections also appear to be an emerging phenomenon in some European countries (32). Coinciding with this trend, treatment of E. faecium infections has increasingly been hampered by the spread of strains resistant to aminoglycosides, β-lactams, and glycopeptides, the three major classes of drugs of therapeutic choice. Lately, E. faecium strains resistant to other classes of newly developed antibiotics have also emerged (17), narrowing available therapeutic options.

We and others recently analyzed the genome sequence of endocarditis-derived E. faecium strain TX0016 and identified 22 genes, named fms (E. faecium surface protein-encoding) genes, predicted to encode LPXTG family cell wall-anchored surface proteins (13, 28). We subsequently identified 15 of the 22 genes, including acm (fms8), a previously described gene encoding a collagen adhesin, as harboring characteristic features of MSCRAMMs and/or pili, such as predicted structural organization into modules with immunoglobulin-like folding, and demonstrated that all 15 genes occurred frequently among 30 clinically derived E. faecium isolates studied (24, 28). Using a larger collection of diverse E. faecium isolates, we detected production of Acm predominantly in isolates derived from clinical sources (23), despite the gene being present in almost all isolates. Furthermore, deletion of acm resulted in significant attenuation in an experimental endocarditis model (22), indicating involvement in E. faecium pathogenesis. Another MSCRAMM-encoding gene that we have characterized, fms10 (subsequently renamed scm), encodes a second collagen-binding MSCRAMM in E. faecium. Recombinant Scm differs from Acm in collagen type specificity by showing affinity for collagen type V instead of types I and IV, which are specifically bound by Acm. Eleven of the remaining 13 genes were found to be clustered into four separate genomic loci, each with an adjacent class C sortase gene characteristic of pilus-encoding genes of gram-positive bacteria, and were predicted to form four distinct pili (28). One of these clusters, E. faecium ebpABC (ebpABCfm), is transcribed as a single operon, and we demonstrated polymerization of its predicted major pilus subunit protein, EbpCfm, into a high-molecular-weight complex (28). A recent study by Hendrickx et al. showed that antibodies against EbpCfm (also referred to as PilB) and Fms21 (also referred to as PilA) stained pili on the surface of E. faecium (12).

Gene association studies have suggested that acquisition of resistance determinants to antibiotics, in particular to ampicillin and subsequently vancomycin, as well as acquisition of other traits, such as a functional acm gene, espEfm (encoding an enterococcal surface protein), and hylEfm (hyaluronidase-like gene), may have facilitated colonization, infection, and/or transmission, although the order of these events cannot currently be determined (16, 23, 26). Hendrickx and colleagues recently demonstrated enrichment of 5 of 22 genes encoding cell wall-anchored proteins in a major hospital-associated genogroup, designated clonal complex 17 (CC17) (16, 34), in a study of 131 E. faecium isolates (12, 13). Hence, it is reasonable to hypothesize that some of the 14 MSCRAMM- and/or pilus-encoding genes (in addition to acm) may also contribute to pathogenesis and/or provide E. faecium with a selective advantage in hospital settings, for example, by conferring adherence properties on host tissues. Enrichment of these traits may also have contributed to the recent nosocomial success of CC17.

In the present study, we tested a large collection of E. faecium isolates to assess the global distribution of the recently identified 14 MSCRAMM- and/or pilus-encoding genes in various clinical and natural populations of E. faecium. The gene distribution and association of the isolates with a clinical or nonclinical origin were also analyzed.

MATERIALS AND METHODS

Bacterial isolates, species identification, and growth conditions.

A total of 433 clinical and nonclinical E. faecium isolates were included in this study. These isolates were collected between 1973 and 2005 from diverse geographic locations (the United States, Argentina, Brazil, France, Belgium, Poland, The Netherlands, Spain, Norway, and China), including epidemic and outbreak strains from around the world (1, 2, 4, 6, 7, 9-11, 15, 18, 19, 23, 26, 27, 29, 31). Isolates known to be identical by pulsed-field gel electrophoresis to other isolates in this collection were excluded. The E. faecium isolates were divided into different groups based on their source of isolation. Among the 264 human clinical isolates (clinical group), 15 were derived from blood cultures of endocarditis patients (endocarditis subgroup) and 57 were from blood cultures of nonendocarditis patients (blood subgroup). The remaining 192 E. faecium clinical isolates, which were collected from specimens of bile, bone, catheters, cervix, cerebrospinal fluid, placenta, peritoneal fluid, sputum, urine, and wounds, were grouped as other clinical isolates (OCI subgroup). Sources of nonclinical isolates included stools of hospitalized patients (n = 69; hospital stool group) and community volunteers (n = 70; community stool group) and animals or animal products (n = 30; animal group). Forty-three of these isolates were previously studied by multilocus sequence typing and/or pulsed-field gel electrophoresis and assessed for the presence of the purK1 allele (a marker for CC17), resulting in 26 being identified as belonging to CC17 and 17 being identified as non-CC17 isolates (23).

All of the E. faecium isolates, initially identified to the species level by biochemical tests, were confirmed by high-stringency colony hybridization using intragenic efafm and aac(6′)-Ii probes, as previously described (9, 29). The E. faecium isolates were grown on brain heart infusion (Difco Laboratories, Detroit, MI) broth/agar and incubated overnight at 37°C.

MSCRAMM- and/or pilus-encoding DNA probes.

Genomic DNA of the sequenced E. faecium strain TX0016 was extracted using a DNeasy blood and tissue kit (Qiagen Inc., Valencia, CA); this DNA was used for PCR amplification of intragenic regions of the 14 MSCRAMM- and/or pilus-encoding genes, using primers listed elsewhere (28). Radiolabeled DNA probes were prepared from the purified PCR products with the RadPrime DNA labeling system (Invitrogen, Carlsbad, CA) according to the protocol supplied by the manufacturer. Unincorporated labeled nucleotides were removed using Micro Bio-Spin P-30 columns (Bio-Rad, Hercules, CA).

Colony hybridization.

Colony lysate membrane preparation and hybridization conditions for enterococci were described in our previous studies (5, 29). In brief, E. faecium isolates were inoculated onto sterile nylon membranes (GE Healthcare, Piscataway, NJ) placed on brain heart infusion agar plates. The sequenced strain E. faecium TX0016 was included as a positive control. After overnight growth at 37°C, colonies were lysed on the membrane after treatment with lysozyme (10 mg/ml) and mutanolysin (4 units/ml). Genomic DNA on the membrane was then denatured, washed, and fixed. Hybridizations were carried out under high-stringency conditions (hybridization buffer containing 50% formamide, 5× Denhardt's solution, 5× SSC [1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.2], 0.1% sodium dodecyl sulfate [SDS], and 100 μg/ml calf thymus DNA) at 42°C overnight, followed by three washes with 2× SSC and 0.1% SDS at room temperature (15 min each) and two washes with 0.1× SSC and 0.1% SDS at 50°C (15 min each).

Statistical analysis.

The distributions of the 14 MSCRAMM- and/or pilus-encoding genes in E. faecium isolates originating from different clinical or nonclinical groups/subgroups were compared using two-tailed Fisher's exact test. P values of <0.05 were considered statistically significant.

RESULTS

Overall distribution of predicted MSCRAMM- and/or pilus-encoding genes in E. faecium isolates.

We and others have shown that acm is present in >99% of isolates of diverse origins (3, 23). The occurrence of the remaining 14 MSCRAMM- and/or pilus-encoding genes among 264 human clinical isolates, 69 stool isolates from hospitalized patients, 70 stool isolates from community volunteers, and 30 animal-derived isolates, collected over a period of 33 years from four continents, is summarized in Table Table1.1. Among all isolates, the percentages of isolates showing hybridization to the individual gene probes ranged from 51% to 98%, but there was substantial variation between different groups, ranging from 67% (fms18) to 99% (fms13) in the clinical group, from 38% (fms18) to 99% (fms13) in the hospitalized patient-derived stool isolate group, from 7% (fms18) to 94% (fms17) in the community volunteer-derived stool isolate group, and from 33% (fms15) to 100% (fms13) in the animal group. The highest frequency of these genes was detected in the endocarditis subgroup, with 11 of the genes being carried by 100% of the isolates. The remaining three genes (fms18, fms20, and fms21) were found in 53%, 80%, and 93% of endocarditis isolates, respectively (Table (Table11).

TABLE 1.
Distribution of 14 predicted MSCRAMM- and/or pilus-encoding genes among 433 temporally and geographically diverse E. faecium isolates

When total numbers of genes were compared between isolates, there were 3,372 genes present of 3,696 possible (91%) among the 264 clinical isolates, 709 genes present of 966 possible (73%) among the 69 stool isolates from hospitalized patients, 597 genes present of 980 possible (61%) among the 70 stool isolates from community volunteers, and 274 genes present of 420 possible (65%) among the 30 isolates from animal or animal products. Pairwise comparisons of these results showed a highly significant difference between the clinical group and each of the three nonclinical groups (P ≤ 0.0001; Fisher's exact test).

Distributions of different combinations of the 14 genes.

As shown in Fig. Fig.1,1, larger numbers of the MSCRAMM- and/or pilus-encoding genes were found in clinical than in nonclinical isolates. As anticipated, stool isolates from hospitalized patients were found to carry a smaller number of the 14 genes than isolates from the clinical group but a larger number than isolates from the two nonclinical groups; this was likely due to fecal samples from hospitalized patients consisting of a mixture of both nosocomially derived and community-derived organisms.

FIG. 1.
Percentages of isolates in different clinical and nonclinical groups that were positive for one or more of the 14 fms genes.

Further examination of the hybridization profiles showed that the presence/absence of the 14 fms genes resulted in 82 observed gene combinations among the 433 isolates. The three most common combinations accounted for 57% of all isolates. More specifically, over one-half of the isolates either had all 14 genes present (28%) or were missing just fms18 (15%) or fms20 (14%). The remaining combinations were more evenly distributed among different isolates. The clinical subgroups contained most of the isolates carrying all or 13 of the genes (81% of clinical isolates), whereas such isolates were not found in the community stool group and were relatively rarely found (13%) in the animal group (Table (Table2).2). Furthermore, over two-thirds (70%) of the community stool isolates carried only 8 to 11 of the 14 genes studied.

TABLE 2.
Distribution of E. faecium isolates based on the number of genes present

Comparison of the distribution of each MSCRAMM- and/or pilus-encoding gene in infection-derived E. faecium isolates versus that in community isolates or animal isolates. (i) Distribution of the 11 MSCRAMM- and/or pilus-encoding genes clustered at four different genomic loci of TX0016. (a) ebpfm operon genes.

The ebpAfm, ebpBfm, and ebpCfm (pilB) genes were found to coexist, consistent with our previous demonstration of transcriptional expression of this gene cluster as a single operon (Table (Table1)1) (28). The ebpABCfm operon was found in all but a few clinical isolates (96 to 100%) but was less frequently (73 to 77%) found in nonclinical isolates (community and animal isolates). There was a significant difference for each of the three clinical subgroups versus the community fecal group (P = 0.019 to <0.0001) and for the blood and OCI subgroups versus animal group isolates (P = 0.0070 and 0.0012, respectively).

(b) fms11-fms19-fms16 cluster.

Similar to the ebpABCfm operon and in agreement with our earlier genome analysis predicting the organization of the fms11-fms19-fms16 cluster as an operon, all three genes were carried by each of the isolates that was positive for any one of the genes (Table (Table1).1). These three genes were present in most isolates of the three clinical subgroups (ranging from 90 to 100%) but were less common among community (43%) and animal (47%) isolates, presenting a highly significant statistical difference when each of the clinical subgroups was compared against either the community or animal isolates (P = 0.0002 to 0.0001).

(c) fms14-fms17-fms13 cluster.

Although the majority of isolates in all groups carried the fms14-fms17-fms13 cluster (Table (Table1)1) (96% of clinical isolates, 83% of community stool isolates, and 73% of animal isolates), there was still a significant difference between two of the clinical subgroups (blood isolates and OCI) and the community stool or animal group (P = 0.0002 to 0.038). However, although analysis of the TX0016 genome predicted organization of this cluster as a pilus-encoding operon, 10% (45/433 isolates) of isolates did not hybridize to one or two of the gene probes of this cluster while hybridizing to the other probe(s). Most (35 [78%]) of these discordant isolates were derived from nonclinical sources.

(d) fms21-fms20 cluster.

Two other genes were also found in very close proximity to each other in the genome of TX0016, namely, fms20 and a major pilus subunit-encoding gene, fms21 (pilA) (12, 13, 28). However, they are separated by a putative class C sortase gene and another open reading frame that lacks a cell wall anchoring motif. The majority of all clinical isolates (91%) carried fms21, while fms20 was less common than fms21 and the second least common gene overall (after fms18); fms20 and fms21 genes coexisted in only 66% of the 433 isolates.

(ii) Distribution of the three fms genes not clustered with other MSCRAMM- and/or pilus-encoding genes of TX0016.

As shown in Table Table1,1, the nonclustered MSCRAMM-encoding genes of the sequenced TX0016 genome (scm, fms15, and fms18) showed various distributions in different groups, albeit with a considerably higher frequency of each gene in each of the three clinical subgroups than in the community-derived stool isolate group. No significant variation between the different clinical subgroups (endocarditis isolates, blood isolates, and OCI) was observed (Table (Table1).1). It is notable that carriage of fms18 was less frequent in the different groups than that of all other genes. Although there was a clear-cut association between the presence of scm and a clinical origin (97% of clinical isolates versus 46% of community-derived isolates; P < 0.0001), the scm gene was also overrepresented in isolates of animal origin (93%). In contrast, fms15 and fms18 were detected in a minority of animal isolates, and the difference between animal isolates and each of the three clinical subgroups was highly significant for fms15 (P < 0.0001), as was the difference between animal isolates and both the blood and OCI subgroups for fms18 (P = 0.0004 to 0.004).

Presence of the 14 genes in CC17.

We previously analyzed 43 isolates studied here for their ancestral relationships and identified 26 of them as CC17 isolates and 17 as non-CC17 isolates (23). Among these, 11 of the 14 genes were present in 100% of the CC17 isolates, while the remaining three genes, fms18, fms21, and fms20, were present in 58%, 92%, and 81% of the CC17 isolates, respectively (Table (Table3).3). A widely variable but generally less frequent occurrence was detected in non-CC17 isolates, ranging from 29% to 100%. Overall, 9 of the 14 genes showed a significantly higher carriage in CC17 isolates than in non-CC17 isolates, while no statistically significant enrichment among CC17 isolates was observed for five of the genes.

TABLE 3.
Comparison of gene distributions of CC17 and non-CC17 isolates

DISCUSSION

The ability of bacteria to infect and cause disease is often dependent on specific genes that distinguish more pathogenic strains from their less pathogenic relatives. In gram-positive organisms, such as streptococci and staphylococci, these virulence-associated determinants include surface-anchored MSCRAMMs (e.g., collagen binding protein Cna of Staphylococcus aureus), surface pili (e.g., fibronectin-collagen-T antigen-carrying pili of Streptococcus pyogenes), secreted proteins or enzymes (e.g., gelatinase of E. faecalis), genes involved in the synthesis of toxins (e.g., Panton-Valentine leukocidin genes of S. aureus), and capsular exopolysaccharides (e.g., the Streptococcus pneumoniae capsule). Recent reports, including one from our laboratory, have assessed the distribution of newly identified or predicted surface protein genes, including MSCRAMM- and/or pilus-encoding genes, in various groups of E. faecium isolates, with each subgroup consisting of relatively few isolates that were collected from various human and animal sources (12, 13, 28). Results from these studies showed that eight MSCRAMM- and/or pilus-encoding genes (ebpAfm, ebpBfm, ebpCfm, scm, fms13, fms15, fms17, and fms21) were widely distributed and present in 82 to 94% of isolates; the remaining six genes were found in 26 to 56% isolates, while acm was previously shown to be present in >99% of isolates (23).

To determine the presence of these 14 MSCRAMM- and/or pilus-encoding genes in various E. faecium isolates from clinical and nonclinical environments, we employed an expanded collection of 433 geographically and temporally diverse E. faecium isolates. Analysis of the total number of these 14 genes in each group showed a considerably higher carriage rate (91%) for the clinical group than for the community stool and animal groups (61% and 65%, respectively), indicating an overall accumulation and/or higher level of preservation of these genes in isolates associated with infections in humans. A similar conclusion can be derived from the hybridization data for 131 isolates recently presented by Hendrickx et al. (12, 13) in which an estimated 88% of the total number of possible genes were carried by clinical and hospital outbreak-associated isolates and 56 to 76% of possible genes were carried by isolates from other sources. This is in contrast to acm, which was carried by >99% of all isolates, although the Acm protein was expressed almost exclusively by clinical isolates and was often present as a pseudogene in nonclinical isolates (23).

Independent analysis of the distribution of each of the genes studied here also pointed to a strong association with a clinical source of isolation for three nonclustered genes (scm, fms15, and fms18) and two of the four pilus-encoding clusters (ebpABCfm and fms11-19-16). Taken together, these nine genes were significantly enriched in each of the three clinical subgroups compared to the community stool group. With the exception of scm, these genes also occurred significantly more often in two or more clinical subgroups than in the animal group. When the clinical subgroups were combined and compared with the community stool and/or animal group, a significant association with clinical origin was also observed for four additional genes (fms14, fms17, fms13, and fms21). Although statistical analyses confirmed a significant enrichment of these genes in each of the infection-derived groups (P < 0.0001 to 0.019), we acknowledge that it would be an oversimplification to directly infer function from the presence or absence of a given gene, particularly since some of the genes may be nonfunctional, as evidenced by previous findings of acm, fms15, fms16, and fms19 as pseudogenes in some isolates (23, 28). Although direct comparisons with the results of Hendrickx et al. (12, 13) are complicated by the lack of information on the presence of each gene in the different clinical and nonclinical groups, our results generally agree with these reports, which showed similar enrichment of these genes. Subsequent assessment of gene distribution in a subset of our isolates that were previously categorized as either CC17 or non-CC17 isolates showed more enrichment in CC17 isolates than that found by Hendrickx et al. (13), with 11 of the 14 genes present in 100% (26) of CC17 isolates, while the remaining 3 genes were present in 58 to 92% of CC17 isolates. Furthermore, we detected some genes in non-CC17 isolates more frequently, e.g., fms14 and fms20, which were present in 88% and 76% of non-CC17 isolates, respectively, in our collection, compared to 41% and 42% of non-CC17 isolates, respectively, in the study of Hendrickx et al. (13). On the other hand, two of the genes, scm and fms15, were present at lower rates in non-CC17 isolates (65% and 29%, respectively) in our study than the previously published rates of 92% and 77% for non-CC17 isolates (13), but this may be a result of our small non-CC17 isolate sample size.

As shown in Table Table1,1, some of the isolates (mostly from nonclinical groups) did not hybridize to one or two genes of the fms14-17-13 cluster, suggesting the possibility of a high recombination rate in this genomic region, which could lead to either divergent gene sequences or deletion of the genes altogether. Interestingly, the variation in the fms14-17-13 cluster resulted mainly from the absence of fms14 and/or fms17 (39/45 isolates), while the putative pilus backbone subunit gene, fms13, was missing in only 6 of the 45 divergent isolates. Similarly, fms20 was absent in 25% of isolates positive for the predicted major pilin gene, fms21, and rarely (0.5% [2/433 isolates]) existed independently. While we were not able to utilize a low-stringency hybridization approach to detect divergent alleles due to apparent cross-reactivity with other pilus-encoding genes, PCR analyses using multiple intra- and intergenic primer pairs also failed to amplify these gene fragments from the selected isolates tested (data not shown). Similar variant pilus subunit-encoding gene clusters have been reported for other gram-positive organisms (30).

As expected from the individual gene results, larger numbers of genes were found to be carried by clinical isolates than by nonclinical isolates (Fig. (Fig.1).1). Concurrent with this, clinical isolates showed less variability in their gene content, with the majority of them (80% [212/264 isolates]) exhibiting only three different gene combinations of the 82 combinations found among all isolates, while the three most common combinations for the community stool and animal groups accounted for only 34% (24/70 isolates) and 27% (8/30 isolates) of the isolates, respectively. Accumulation of these genes in clinical isolates may be a marker of their clonal origin, a potential factor that influences the distribution of these genes as well as enrichment of specific gene combinations. On the other hand, the considerable variability seen in nonclinical isolates, e.g., the presence of pilus clusters in isolates with otherwise heterogeneous contents of the 14 genes, may have resulted from horizontal gene transfer between isolates that are not closely related evolutionarily.

In summary, our observation of the more frequent occurrence of many of the MSCRAMM- and pilus-encoding genes in clinical isolates than in fecal isolates from community volunteers and, generally, animal isolates suggests that their preservation and/or enrichment may have contributed to the survival and dissemination of E. faecium in hospital environments. Our ongoing characterization of these genes aims at revealing their potential role(s) in E. faecium pathogenesis, such as adherence to host cells and tissues.

Acknowledgments

This work was supported in part by NIH grant R01 AI067861 from the Division of Microbiology and Infectious Diseases, NIAID, to Barbara E. Murray.

Footnotes

[down-pointing small open triangle]Published ahead of print on 4 February 2009.

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