Accessory gene regulator (agr) locus in geographically diverse Staphylococcus aureus isolates with reduced susceptibility to vancomycin

Abstract

The majority of infections with glycopeptide intermediate-level resistant Staphylococcus aureus (GISA) originate in biomedical devices, suggesting a possible increased ability of these strains to produce biofilm. Loss of function of the accessory gene regulator (agr) of S. aureus has been suggested to confer an enhanced ability to bind to polystyrene. We studied agr in GISA, hetero-GISA, and related glycopeptide-susceptible S. aureus isolates. All GISA strains from diverse geographic origins belong to agr group II. All GISA strains were defective in agr function, as demonstrated by their inability to produce delta-hemolysin. Hetero-GISA isolate A5940 demonstrated a nonsense mutation in agrA that was not present in a pulsed-field gel electrophoresis-indistinguishable vancomycin-susceptible isolate from the same patient. Various other agr point mutations were noted in several clinical GISA and hetero-GISA isolates. A laboratory-generated agr-null strain demonstrated a small but reproducible increase in vancomycin heteroresistance after growth in vitro in subinhibitory concentrations of vancomycin. This was not seen in the isogenic agr group II parent strain in which agr was intact. The in vitro bactericidal activity of vancomycin was attenuated in the agr-null strain compared to the parent strain. These findings imply that compromised agr function is advantageous to clinical isolates of S. aureus toward the development of vancomycin heteroresistance, perhaps through the development of vancomycin tolerance.

FIG. 1.

PFGE of SmaI-macrorestricted genomic DNA of isolate pair A6300 (lane 1) and A6298 (lane 2) and isolate pair A5937 (lane 3) and A5940 (lane 4). Each pair was isolated from the same patient.

FIG. 2.

Delta-hemolysin assays. Each panel is marked by the test strain streaked horizontally and beta-hemolysin-producing RN4420 streaked vertically. The left panel shows positive control RN6607 (A) and negative control agr-null RN9120 (B). The short arrow denotes the beta-hemolysin of RN4420, and the long arrow demonstrates the enhanced zone of hemolysis created by the interaction of the beta-hemolysin of RN4420 and the delta-hemolysin of the test strain. Note that RN9120 retains the ability to produce beta-hemolysin. The center panel shows A6222 (C), HIP5836 (D), RN6607 positive control (E), Mu3 (F), and Mu50 (G). The right panel shows hetero-GISA A5940 (I) and the related vancomycin-susceptible strain A5937 (H).

FIG. 3.

RFLP analysis with RsaI of an amplified portion of the agr locus of GISA and hetero-GISA strains with diverse geographic origins (indicated in parentheses). Lanes: 1, PC-3 (New York, N.Y.); 2, HIP5836 (New Jersey); 3, A5840 (St. Louis, Mo.); 4: A6298 (Boston, Mass.); 5, A6222 (Boston, Mass.); 6, Mu3 (Japan); 7, SA32 (agr group I). Molecular weight markers displayed are 200 to 800 bp increasing at 200-bp intervals.

FIG. 4.

(A) Translated protein sequences of agrA from GISA and related non-GISA agr group II strains. Differences in amino acids from the consensus are boxed. (B) Translated protein sequences of agrB. Differences from prototype agr group II strain SA502A are shown in boxes. (C) Translated protein sequences of agrC. Differences from prototype agr group II strain SA502A are shown in boxes. Sequences for SA502A, N315, and Mu50 were derived from GenBank as referenced in the text. Note that the sequence of agrC of SA502A was modified after communication with the authors of the original sequence as stated in the text (16).

FIG. 4.

(A) Translated protein sequences of agrA from GISA and related non-GISA agr group II strains. Differences in amino acids from the consensus are boxed. (B) Translated protein sequences of agrB. Differences from prototype agr group II strain SA502A are shown in boxes. (C) Translated protein sequences of agrC. Differences from prototype agr group II strain SA502A are shown in boxes. Sequences for SA502A, N315, and Mu50 were derived from GenBank as referenced in the text. Note that the sequence of agrC of SA502A was modified after communication with the authors of the original sequence as stated in the text (16).

FIG. 4.

(A) Translated protein sequences of agrA from GISA and related non-GISA agr group II strains. Differences in amino acids from the consensus are boxed. (B) Translated protein sequences of agrB. Differences from prototype agr group II strain SA502A are shown in boxes. (C) Translated protein sequences of agrC. Differences from prototype agr group II strain SA502A are shown in boxes. Sequences for SA502A, N315, and Mu50 were derived from GenBank as referenced in the text. Note that the sequence of agrC of SA502A was modified after communication with the authors of the original sequence as stated in the text (16).

FIG. 5.

(A) Population analyses of agr group II strain RN6607 before (RN6607) and after (RN6607-V) growth in subinhibitory concentrations of vancomycin. (B) Same experiment as in panel A but performed with the agr::tetM isogenic agr-null strain RN9120 before (RN9120) and after (RN9120-V) growth in subinhibitory concentrations of vancomycin.

FIG. 6.

(A) Biofilm assay as determined by the ability to bind polystyrene of RN6607 and RN9120 in TSB, TSB supplemented with 1% glucose (Glu), and TSB supplemented with 4% ethanol (EtOH). The values reported are the mean OD₅₄₀. Christensen et al. classified strains with various ODs obtained by this method as follows: OD < 0.120, nonadherent; OD 0.120 to 0.240, weakly adherent; and OD > 0.240, strongly adherent (7). (B) Bactericidal assay of RN6607 and RN9120 in BHI broth with vancomycin at 16 μg/ml. The lower limit of detection in this assay was at 1.6 log₁₀ CFU/ml.