A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen - PubMed (original) (raw)

A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen

Sreedhar R Nallapareddy et al. Infect Immun. 2008 Sep.

Abstract

Enterococcus faecium recently evolved from a generally avirulent commensal into a multidrug-resistant health care-associated pathogen causing difficult-to-treat infections, but little is known about the factors responsible for this change. We previously showed that some E. faecium strains express a cell wall-anchored collagen adhesin, Acm. Here we analyzed 90 E. faecium isolates (99% acm(+)) and found that the Acm protein was detected predominantly in clinically derived isolates, while the acm gene was present as a transposon-interrupted pseudogene in 12 of 47 isolates of nonclinical origin. A highly significant association between clinical (versus fecal or food) origin and collagen adherence (P <or= 0.0003) was also demonstrated, and levels of adherence were highly correlated (r = 0.879) with the amount of cell surface Acm detected by whole-cell enzyme-linked immunosorbent assay and flow cytometry. Thirty-seven of 41 sera from patients with E. faecium infections showed reactivity with recombinant Acm, while only 4 of 30 community and hospitalized patient control group sera reacted (P <or= 0.0003); importantly, antibodies to Acm were present in all 14 E. faecium endocarditis patient sera. Although pulsed-field gel electrophoresis indicated that multiple strains expressed collagen adherence, multilocus sequence typing demonstrated that the majority of collagen-adhering isolates, as well as 16 of 17 endocarditis isolates, are part of the hospital-associated E. faecium genogroup referred to as clonal complex 17 (CC17), which has emerged globally. Taken together, our findings support the hypothesis that Acm has contributed to the emergence of E. faecium and CC17 in nosocomial infections.

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Figures

FIG. 1.

Adherence of E. faecium isolates to immobilized collagen type I. Data points for 90 isolates in this study represent mean percentages of cells adhering ± standard deviations; data for 30 additional previously reported E. faecium nonlaboratory isolates (20) are shaded in gray. Isolates were considered to adhere if ≥5% of total labeled cells adhered to collagen coating the well (34).

FIG. 2.

Correlation between collagen adherence and surface display of Acm by 31 E. faecium strains (11 endocarditis isolates, 10 Other-Clin isolates, and 5 isolates from each of the nonclinical groups). Adherence percentages were plotted as a function of OD450 values from a whole-cell ELISA using E. faecium strains and affinity-purified anti-rAcm A-specific antibodies (P < 0.0001 for correlation).

FIG. 3.

Quantitation of Acm surface expression by fluorescence-activated cell sorting analysis. (A to D) Analysis of four endocarditis-derived collagen-adhering E. faecium isolates exhibiting different levels of collagen adherence. Percentages of radiolabeled cells adhering to collagen were 43%, 36%, 11%, and 6% for TX0074, TX2535, TX0082, and TX2658, respectively. (E to J) Analysis of five non-collagen-adhering E. faecium endocarditis isolates and an acm deletion mutant of TX0082. Percentages of radiolabeled cells adhering to collagen were 2.3%, 2.7%, 3.9%, 2.3%, and 2.2% for TX0016, TX0068, TX0080, TX0110, and TX0111, respectively. Bacteria were analyzed by flow cytometry, using side scatter as the threshold for detection. Specific binding by anti-Acm antibodies is indicated as log fluorescence intensity on the x axis. Each histogram represents 50,000 events (bacterium-sized particles). PI, preimmune Igs; anti-Acm, anti-Acm A-domain-specific Igs.

FIG. 4.

PCR detected acm disruptions in 14 different E. faecium isolates. (A) Schematic representation showing the positions of disruptions by different IS elements analyzed in isolates of this study and a previous study (20). RBS, predicted ribosomal binding site; S, signal peptide; A domain, nonrepetitive collagen binding domain; B domain, domain with variable number of repeats in different strains; W, cell wall domain with motif required for sortase-mediated surface anchoring; M, membrane-spanning domain; and C, charged C-terminal domain. (B) Distribution of disrupted acm genes among 89 _acm_-positive E. faecium isolates from this study. †, the four collagen-adherent and two nonadherent endocarditis isolates of our previous study included in this study for molecular epidemiological analyses and surface expression level studies also had an uninterrupted acm gene (20); *, a single isolate of animal origin lacked acm.

FIG. 5.

IS_256_-mediated modulation of acm expression in E. faecium strain TX2466. (A) Enhanced collagen adherence of acm pseudogene-containing E. faecium isolate TX2466 after growth in collagen-supplemented BHI broth. (B) Fluorescence-activated cell sorting analysis of TX2466 grown in BHI or grown in wells coated with collagen. PI, preimmune Igs; anti-Acm, anti-Acm A-domain-specific Igs. (C) Agarose gel electrophoresis of colony PCR amplification with primers AcmF1 and AcmR4, using a mixture of cells before and after repeated passages in wells coated with immobilized collagen. (D) Schematic representation of the IS_256_ insertion site and its precise excision, confirmed by sequencing of the acm gene of TX2466. The duplicated target sequences of IS_256_ transposase are boxed.

FIG. 6.

Reactivity of human serum with rAcm A protein. (A) Immunoblots of rAcm A protein probed with human sera. Lanes 1, 2, and 4 to 6, sera from different patients with E. faecium endocarditis; lane 3, molecular size standards; lanes 7 to 14, NHS obtained from healthy volunteers. (B) Distribution of anti-Acm A domain Ig titers in human sera. The four endocarditis patient sera that had antibodies to Acm but whose infecting strain lacked in vitro collagen adherence and surface Acm are shaded in black. Sera from hospitalized patients as well as healthy volunteers were used as two groups of controls.

FIG. 7.

Analysis of 23 E. faecium isolates by PFGE. (A) SmaI PFGE profiles of PF1, PF2, and other strains. Six endocarditis, one catheter, and one wound isolate, isolated between 1992 and 2002 from several hospitals in Texas, were categorized as PF1. Two endocarditis isolates from Arizona and Texas were categorized as possibly related (PF2). (B) SmaI PFGE profiles of strains with distinct PFGE patterns. Nonendocarditis isolates belonging to the Other-Clin group are marked with the symbol “§”.

References

1. Boyce, J. M., S. M. Opal, J. W. Chow, M. J. Zervos, G. Potter-Bynoe, C. B. Sherman, R. L. Romulo, S. Fortna, and A. A. Medeiros. 1994. Outbreak of multidrug-resistant Enterococcus faecium with transferable vanB class vancomycin resistance. J. Clin. Microbiol. 321148-1153. -PMC -PubMed
1. Camargo, I. L., M. S. Gilmore, and A. L. Darini. 2006. Multilocus sequence typing and analysis of putative virulence factors in vancomycin-resistant and vancomycin-sensitive Enterococcus faecium isolates from Brazil. Clin. Microbiol. Infect. 121123-1130. -PubMed
1. Cereda, R. F., A. C. Gales, S. Silbert, R. N. Jones, and H. S. Sader. 2002. Molecular typing and antimicrobial susceptibility of vancomycin-resistant Enterococcus faecium in Brazil. Infect. Control Hosp. Epidemiol. 2319-22. -PubMed
1. CLSI. 2003. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, 6th ed. Document M7-A6. Clinical and Laboratory Standards Institute, Wayne, PA.
1. CLSI. 2005. Performance standards for antimicrobial susceptibility testing, 15th informational supplement. Document M100-S15. Clinical and Laboratory Standards Institute, Wayne, PA.

A functional collagen adhesin gene, acm, in clinical isolates of Enterococcus faecium correlates with the recent success of this emerging nosocomial pathogen - PubMed (original) (raw)