Differential Abilities of Capsulated and Noncapsulated Staphylococcus aureus Isolates from Diverse agr Groups To Invade Mammary Epithelial Cells

Bacterial isolates and growth conditions.One hundred twenty-six epidemiologically unrelated S. aureus isolates were obtained from the milk of cows with mastitis from herds located in different districts of Argentina (31). The genetic relationship among most of these isolates was assessed previously by SmaI pulsed-field gel electrophoresis typing and automated EcoRI ribotyping (4). Analysis of 17 strains included in internalization assays was performed by arbitrarily primed PCR (AP-PCR) (36) and STAR restriction profile (STAR-RP) analyses (26). Genotypes defined by AP-PCR were identified by a single lowercase letter in order to simplify description of the results. All strains investigated were susceptible to methicillin. S. aureus was identified by a standard procedure of the bacteriology laboratory (2). All bacteria were stored in trypticase soy broth (Difco, Detroit, MI) medium with 20% glycerol at −20°C until use. For the experiments, bacterial cells were collected by centrifugation, washed with sterile saline solution, and suspended in invasion medium (see below) to a density of ca. 10⁷ CFU/ml. Production of CP5 and CP8 by clinical strains and their agr-null mutants was assessed by a colony immunoblot assay (16) and confirmed by an immunodiffusion test (33). None of the S. aureus strains utilized in this study produced mucoid colonies on Columbia salt agar. Isolates not reactive with antibodies to CP type 1, 2, 5, or 8 were defined as nontypeable (NT) (5). The agr mutant (Δagr::tetM) was transduced using φ11 lysates of the original agr mutant RN6911 into bovine strains RA19 and RA8. Transductants did not show hemolysis on rabbit and sheep blood agar and did not produce RNAIII, as assessed by reverse transcription-PCR.

Genomic DNA extraction.Chromosomal DNA was purified from bovine S. aureus isolates after bacterial lysis with lysostaphin (5 μg/ml) and lysozyme (10 μg/ml) (Sigma Chemical Co., St. Louis, MO) by the method of Pitcher et al. (23).

Multiplex PCR.The agr groups were determined by a multiplex PCR described previously by Gilot et al. (7). Briefly, purified nucleic acids (1 ng/μl) were amplified in a 25-μl reaction mixture containing 0.25 U/μl of Taq DNA polymerase (Invitrogen Corp., CA), 200 μM deoxynucleoside triphosphates (Promega, Madison, WI), 5 mM MgCl₂, and the following primers (0.3 μM): Pan (5′-ATG CAC ATG GTG CAC ATG C-3′), agr1 (5′-GTC ACA AGT ACT ATA AGC TGC GAT-3′), agr2 (5′-TAT TAC TAA TTG AAA AGT GGC CAT AGC-3′), agr3 (5′-GTA ATG TAA TAG CTT GTA TAA TAA TAC CCA G-3′), and agr4 (5′-CGA TAA TGC CGT AAT ACC CG-3′). Multiplex PCRs were performed in an Eppendorf thermal cycler (Mastercycler Gradient) for 1 cycle at 94°C for 1 min; 26 cycles at 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min; and finally 1 cycle at 72°C for 10 min. Amplification products were subjected to electrophoresis in a 1.5% agarose gel containing ethidium bromide and visualized by transillumination under UV.

Restriction fragment length polymorphism of the agr variable region.Amplification of the agr locus variable region by PCR was performed using primers B1 (5′-TAT GCT CCT GCA GCA ACT AA-3′) and C2 (5′-CTT GCG CAT TTC GTT GTT GA-3′) as described by van Leeuwen et al. (37). Genomic DNA was amplified in a 100-μl reaction mixture containing Taq DNA polymerase (2.5 U) (Invitrogen), 200 μM deoxynucleoside triphosphates, 0.5 μM primers, and 2.2 mM MgCl₂. Reactions were performed for 1 cycle at 94°C for 4 min; 40 cycles at 94°C for 1 min, 50°C for 1 min, and 74°C for 2 min; and finally 1 cycle at 74°C for 3 min. Samples were stored at −20°C before restriction. Then, agr amplicons (1,070-bp variable region of the agr operon) were restricted with RsaI (Promega) and AluI (Invitrogen) according to the manufacturer's instructions. The restriction fragments were separated by electrophoresis on a 3% agarose gel stained with ethidium bromide and visualized by transillumination under UV. agr alleles were defined and recorded according to the restriction patterns obtained with RsaI (capital letters) and AluI (arabic numbers).

Cell culture.The established bovine mammary epithelial cell line (MAC-T) (13) was generously provided by Nexia Biotechnologies (Quebec, Canada). MAC-T cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco BRL), insulin (5 μg/ml), hydrocortisone (5 μg/ml), penicillin (100 U/ml), and streptomycin sulfate (100 μg/ml) (Sigma Chemical Co., St. Louis, MO). Prior to each experiment, MAC-T cells were seeded at 6 × 10⁴ cells/well in 24-well tissue culture plates and grown for 3 days at 37°C with 6% CO₂.

Internalization assays.Confluent MAC-T cell monolayers (approximately 2 × 10⁵ to 2.5 × 10⁵ cells/well) were washed four times with sterile phosphate-buffered saline (PBS) and then inoculated with bacteria suspended in fresh growth medium without antibiotics (invasion medium) to produce multiplicities of infection of 10 to 40. After incubation for 1 h at 37°C under 6% CO₂, the wells were washed with PBS and then 1 ml of invasion medium supplemented with 100 μg of gentamicin (Sigma) was added to each well to kill extracellular bacteria. Incubation of cocultures with gentamicin proceeded for an additional 2 h at 37°C with 6% CO₂. Supernatants were then collected and plated on trypticase soy agar (TSA) to verify the killing by gentamicin. The monolayer was washed four times with sterile PBS, treated for 5 min at 37°C with 100 μl of 0.25% trypsin-0.1% EDTA (Gibco BRL), and lysed by the addition of 900 μl of 0.025% Triton X-100 (USB, Cleveland, OH) in sterile distilled water to release intracellular staphylococci. The CFU number was determined by quantitative plating on TSA. MAC-T cell viability was evaluated by trypan blue exclusion.

Mouse ima infection model.Outbred CF1 mice were bred and kept in the vivarium of the Department of Microbiology, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina. Animals were maintained according to the guidelines set forth by the National Institutes of Health Research Council (20). Seven to 10 days after parturition, groups of lactating female mice were inoculated with 0.05 ml of the bacterial suspension in physiologic saline solution (1 × 10⁶ CFU/gland) by the intramammary (ima) route, as described previously (34). After 1 and 4 days, the left and right fourth (L4 and R4) mammary glands were aseptically removed and homogenized. Viable counts were performed on these homogenates by plating the samples on TSA. The experimental mouse model utilized in this study was recently validated as an experimental approach to the study of bovine mastitis (3).

Statistical analysis.Multiple comparisons of intracellular-CFU/ml counts between agr groups were performed by the Kruskal-Wallis test. Nonparametrical data were analyzed with the Mann-Whitney test using GraphPad software (version 2.2; PRISM). P values lower than 0.05 were considered significant.

agr group of interference and polymorphism.Multiplex PCR was performed using primers specific for the agrB, agrD, and agrC genes, as described in Materials and Methods. One hundred twenty-six epidemiologically unrelated bovine S. aureus strains were analyzed as clone representatives from Argentina. Most strains isolated from bovines with mastitis belonged in agr group I (88%). Eight percent of the strains were identified as being in agr group III, whereas the remainder was classified in agr groups II and IV (2 and 2%, respectively). All S. aureus agr strains of types II, III, and IV expressed CP8, whereas agr group I S. aureus isolates were either CP5 or NT (Table 1). One hundred five of 111 agr group I isolates were representative of subtypes (>80% similitude) from a single isolate cluster found in Argentina (4). The polymorphism of the agr locus was determined by restriction profile analysis after amplification of the variable region (1,070 bp) of the agr operon. Combination of the AluI and RsaI restriction patterns permitted identification of six agr restriction types. All agr group I strains belonged in prevalent allele A/1, whereas the strains from group II (B/2 and B/3), group III (C/5 and D/5), and group IV (B/2 and D/4) exhibited more diversity.

agr group and MAC-T cell invasion.The influence of the agr group on the ability of S. aureus to remain within host cells was evaluated by the epithelial MAC-T cell invasion assay. Two hours after addition of gentamicin, the numbers of intracellular CFU in agr group I strains were significantly higher than those in group II, III, and IV strains (Fig. 1) . Molecular epidemiologic analysis of the 17 strains mentioned in Fig. 1 revealed 13 genotypes with less than 80% homology (ascertained as described in Materials and Methods) and that no single S. aureus clone was associated with any agr or capsule serotype group. The agr system was probably active in 14 of the 17 strains tested since they were able to express CP. One of the strains (RA17) expressing neither CP5 nor CP8 (NT) exhibited a total deletion of the cap cluster and a concomitant insertion of an IS257 variant (5). The other two strains (RA19 and RA20) exhibited positive amplification for capH-J5. The many reasons why S. aureus isolates bearing apparently conserved cap5 or cap8 clusters fail to express capsules has been described recently. In any event, production of the RNAIII transcript was detected in these three strains (Fig. 1), as ascertained by reverse transcription-PCR, suggesting that the agr system was also functional in strains RA17, RA19, and RA20. Therefore, increased internalization may not be due to any functional deficiency of the agr system. It must be noted that more than one-half of the S. aureus NT isolates from Argentina carry the IS257-mediated deletion of the cap gene cluster (35). Overall, agr group I strains are more efficiently internalized than strains of any other agr type. The statistical significance of this phenomenon is not affected by CP expression, since the internalization of any agr group I CP5 S. aureus isolate was significantly increased compared to that of any isolate in any remaining agr group (Fig. 1). Figure 1 shows data for isolates representative of the population under scrutiny (31), which exhibits the distinctive feature of exhibiting neither CP5 isolates within agr groups II, III, and IV nor CP8 isolates within agr group I. Furthermore, all NT isolates in this population belong in agr group I (Table 1).

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FIG. 1.

agr groups and S. aureus invasion of the mammary epithelial cell. Each bar represents the arithmetic mean ± standard error of the mean for numbers of intracellular CFU/ml for individual strains for 5 to 10 independent experiments. Multiple comparisons between numbers of intracellular CFU/ml from agr groups I, II, III, and IV revealed statistically significant differences (P < 0.0001, Kruskal-Wallis test). The stars represent significant differences between each one of the agr II, III, and IV isolates and the lowest bar of the agr group I CP5 strain (eighth bar from the left). Significant differences differed, with P values ranging from 0.04 to 0.0004 (Mann-Whitney test). The genotype for each strain is as follows (1 to 17 from left to right): 1, d; 2, j; 3, j; 4, l; 5, l; 6, m; 7, k; 8, f; 9, c; 10, e; 11, g; 12, i; 13, h; 14, h; 15, b; 16, a; and 17, b. Four pairs of isolates were not discriminated by the methods utilized. However, isolates 15 and 17 were obtained from different subregions of Argentina 9 years apart and isolates 13 and 14 were obtained from different subregions 2 years apart. Isolates 4 and 5 were obtained from the same subregion but differed in STAR-RP pattern, whereas isolates 2 and 3 were obtained from the same subregion 9 months apart.

Two pairs of isogenic agr group I strains were included in the experiments in order to test whether impairment of the agr system in a CP5 (RA8) or an NT (RA19) background affects the internalization of S. aureus bacteria into MAC-T cells. To this purpose, agr-null mutants of strains RA8 and RA19 were obtained as described in Materials and Methods. As expected, the agr mutant derived from RA8 showed an NT phenotype. RA8 and its agr-null derivative showed a significant increase in the number of intracellular agr-null S. aureus mutants compared with the wild-type strain (Fig. 2). This result may be partially attributed to loss of capsule expression by the agr-null mutant. However, a significant increase in the number of intracellular CFU of the agr-null RA19 strain was also observed when this strain was compared with the agr group I parental strain RA19 (both NT) (Fig. 2). Moreover, the agr-null RA19 mutant was internalized into MAC-T cells in significantly greater numbers than the agr-null RA8 mutant. These results demonstrate that the increased internalization of agr group I strains may be due not only to loss of capsule expression but also to particular features of the genetic background which may contain other genes up- or down-regulated by the agr group I system.

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FIG. 2.

MAC-T cell invasion by S. aureus agr-null mutants. MAC-T cells were inoculated with agr I group S. aureus RA8 (striped bars) and RA19 (gray bars) strains and with their respective agr-null mutants for internalization assays. Each bar represents the arithmetic mean ± standard error of the mean for numbers of intracellular S. aureus CFU/ml for five independent experiments. Levels of significance for comparisons of a versus b, c versus d, and b versus d were P values of <0.001 (Mann-Whitney test).

agr group and persistence in vivo.To evaluate the influence of agr types on persistence in vivo, S. aureus strains from each agr group were selected to be evaluated in the mouse mastitis model. Groups of lactating mice were inoculated with the bacterial suspension (1 × 10⁶ CFU/gland) by the ima route. At 1 and 4 days postinoculation, the mammary glands (L4 and R4) were removed and homogenized, and homogenates were plated on TSA to assess the bacterial colonization of the tissue. By days 1 and 4 after inoculation, the CFU total for S. aureus RA19 (agr group I, NT) was significantly higher than those for S. aureus agr groups II and IV (Fig. 3). In order to establish whether the observed differences were solely due to the lack of CP expression by S. aureus RA19, S. aureus RA8 (which expresses CP5 and is in agr group I) was included as a control. Even though there was an increased clearance of the agr group I CP5 S. aureus strain compared with that of the NT S. aureus strain, differences between the capsulated agr group I and the capsulated agr group II and IV strains remained significant (Fig. 3). Moreover, significantly more S. aureus RA19 bacteria (787 CFU/ml) were internalized into MAC-T cells, as measured at 24 h postinfection, than S. aureus RA4 (agr group II, CP8) (14 CFU/ml) and RA10 (agr group IV, CP8) bacteria (3.5 CFU/ml) (P < 0.01, Mann-Whitney test).

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FIG. 3.

agr group and bacterial persistence in the murine mastitis model. Groups of mice were inoculated (1 × 10⁶ CFU/gland) by the ima route with a suspension of bovine S. aureus isolates. At 1 and 4 days postinfection, viable counts were determined. Each point represents the median for numbers of S. aureus CFU/gland for 8 to 16 mammary glands. By day 1, significant differences for strains marked ★ were as follows: for agr group I (RA19, NT) versus agr group II (RA4 and RA7) or agr group IV (RA10 and RA16), P values of <0.01 (Mann-Whitney test); for agr group I (RA8, CP5) versus agr group II (RA4 and RA7) or agr group IV (RA10 and RA16), P values of <0.01 (Mann-Whitney test). By day 4, significant differences for strains marked ★ were as follows: for agr group I (RA19, NT) versus agr group II (RA4 and RA7) or agr group IV (RA10), P values of <0.01 (Mann-Whitney test); for agr group I (RA8, CP5) versus agr group II (RA4), P values of 0.03 (Mann-Whitney test).