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Infection and Immunity, February 2007, p. 886-891, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.01215-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Fernanda R. Buzzola,1 Lucía P. Alvarez,1 Lorena P. N. Tuchscherr,1 María S. Barbagelata,1 Santiago M. Lattar,1 Luis Calvinho,2 and Daniel O. Sordelli1*

Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, C1121ABG Buenos Aires, Argentina,1 Estación Experimental Agropecuaria Rafaela, INTA, CP2300 Santa Fe, Argentina2

Received 1 August 2006/ Returned for modification 11 October 2006/ Accepted 20 November 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Staphylococcus aureus is the bacterium most frequently isolated from milk of bovines with mastitis. Four allelic groups, which interfere with the regulatory activities among the different groups, have been identified in the accessory gene regulator (agr) system. The aim of this study was to ascertain the prevalence of the different agr groups in capsulated and noncapsulated S. aureus bacteria isolated from mastitic bovines in Argentina and whether a given agr group was associated with MAC-T cell invasion and in vivo persistence. Eighty-eight percent of the bovine S. aureus strains were classified in agr group I. The remainder belonged in agr groups II, III, and IV (2, 8, and 2%, respectively). By restriction fragment length polymorphism analysis after PCR amplification of the agr locus variable region, six agr restriction types were identified. All agr group I strains presented a unique allele (A/1), whereas strains from groups II, III, and IV exhibited more diversity. Bovine S. aureus strains defined as being in agr group I (capsulated or noncapsulated) showed significantly increased abilities to be internalized within MAC-T cells, compared with isolates from agr groups II, III, and IV. agr group II or IV S. aureus strains were cleared more efficiently than agr group I strains from the murine mammary gland. The results suggest that agr group I S. aureus strains are more efficiently internalized within epithelial cells and can persist in higher numbers in mammary gland tissue than S. aureus strains classified in agr group II, III, or IV.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Staphylococcus aureus is an important animal and human pathogen responsible for diverse types of severe infections. In animals, S. aureus is the bacterium most frequently isolated from milk of bovines with mastitis (28). Subclinical mastitis represents from 90 to 95% of all cases of bovine udder infection and is usually refractory to antibiotic treatment (21). The limited success of antibiotic therapy may be due to the ability of S. aureus to invade and survive within different cell types found in the mammary gland (1, 12, 29). Therefore, S. aureus can persist in the host for a long time without causing an apparent inflammation and/or clinical infection.

The pathogenesis of S. aureus infection is very complex. The agr (accessory gene regulator) locus is a quorum-sensing system that controls the expression of a variety of genes involved in tissue colonization (e.g., surface proteins) and invasion (e.g., extracellular toxins). Among other virulence factors, capsular polysaccharide (CP) is an important surface component that is up-regulated by agr during the postexponential growth phase (22). The importance of CP to internalization within cells of the infected host is underscored by the finding that reduced or absent CP expression enhances the adherence of S. aureus bacteria to endothelial cells (24). S. aureus specificity agr groups were defined based upon the polymorphisms of agrC, agrD, and agrB (14). There is mutual cross-inhibition between S. aureus isolates from different groups of the agr system (15). Several authors have described distinct agr alleles in S. aureus, as defined by agr restriction fragment length polymorphism, within agr groups (6, 8, 10).

Since a limited number of clones have been found in bovines with mastitis in different regions of the world (4, 11, 33), this study was aimed at ascertaining the prevalence of the different agr groups in S. aureus bacteria isolated from bovines with mastitis in Argentina and whether a given agr group was associated with MAC-T cell invasion and in vivo persistence.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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 ({Delta}agr::tetM) was transduced using {phi}11 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 x 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 x 105 to 2.5 x 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 x 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.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
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.


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  		TABLE 1. agr groups and capsule phenotypes of bovine S. aureus isolates

 
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).


Figure 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.


Figure 2
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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 x 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).


Figure 3
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  		FIG. 3. agr group and bacterial persistence in the murine mastitis model. Groups of mice were inoculated (1 x 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).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The agr system is polymorphic and permits classification of S. aureus strains in four groups. In the present study, it was observed that most bovine S. aureus isolates under scrutiny belonged in agr group I (88%) whereas the remaining strains were evenly distributed among agr groups II, III, and IV. This finding agrees with a previous observation by Gilot and van Leeuwen (8), who reported that 69% of bovine isolates of S. aureus from France (mainly), the United States, the United Kingdom, and Japan belonged in agr group I. Similarly, a study of human S. aureus showed that agr group I was prevalent among clinical strains (6). Several authors have reported the existence of distinct agr alleles within agr groups in S. aureus (19, 32). Amplification of the variable region of the agr locus and subsequent restriction fragment length polymorphism analysis with enzyme RsaI or AluI allowed identification of four (A, B, C, and D) or five (1, 2, 3, 4, and 5) different patterns, respectively. Combination of both restriction profiles allowed definition of six agr alleles in Argentina. The highly prevalent agr group I allele defined in this report as A/1 coincided with the RIII-A1 pattern defined by Gilot et al. (7), which was the most prevalent agr allele and contained 56.3% of the 71 bovine isolates investigated by this author. An investigation on the prevalence of S. aureus from humans and bovines revealed that there is a small, reduced number of prevalent clones of defined agr groups and agr alleles causing disease (8). The same study showed that whereas agr group I S. aureus was highly prevalent in both bovines and humans, there was a distinct allele distribution within the same agr group. The authors concluded that the human and the bovine S. aureus populations are different and that certain alleles in one or the other population became prevalent due to their possession of unique genetic characteristics. In favor of this conclusion, Robinson et al. underscored the importance of host-pathogen interaction governed by agr-mediated virulence gene expression over pathogen-pathogen interactions affected by agr-mediated bacterial interference (27).

The ability of S. aureus to invade and survive in different cell types may contribute to persistence of the bacteria in the bovine mammary gland. In the present study, the bovine S. aureus strains defined as being in agr group I showed increased abilities to invade MAC-T cells. Conversely, isolates of agr groups II, III and IV were internalized less efficiently. Mullarky et al. demonstrated that less frequently isolated S. aureus agr genotypes from bovines were also the genotypes more efficiently attacked by bovine neutrophils (19). In agreement with the authors, we suggest that bovine S. aureus strains defined as being in agr group II, III, or IV may be more susceptible to attack by the host immune response because they tend to remain in larger numbers in the extracellular environment. In fact, agr group II or IV S. aureus strains were cleared more efficiently than agr group I strains from the murine mammary gland. Previous studies have shown that inactivation of the agr system provokes an increased capacity for cell invasion (39). Other authors have found that the agr system is not active during chronic infections (9). As expected, abrogation of the agr system in bovine agr group I S. aureus strains resulted in the absence of CP expression. In addition, the agr-null mutant from capsulated strain RA8 exhibited an increased ability to invade MAC-T cells. Interestingly, the internalization of the acapsular RA19 strain was significantly increased when the strain was converted into an agr-null mutant, and the agr-null RA19 mutant was internalized more efficiently than the agr-null RA8 mutant. In the light of the observations made in the present study, we could speculate that the high capacity of S. aureus agr group I to invade MAC-T cells might be due to specifically suppressed action of the agr locus. However, the agr systems in all bovine S. aureus strains investigated in this study were functional in vitro. Therefore, differences in the degrees of internalization should be attributed to another factor(s). Pragman et al. (25) suggested that under low-oxygen conditions in the S. aureus-infected mammary gland (18), the srrAB system would be activated, inducing down-regulation of the agr system. Under such a condition, capsule production (as well as production of other active exoproducts) would be inhibited and internalization would be favored. Whether the activity of the agr system is relevant to the outcome of infection in the bovine clinical strains of S. aureus is not completely understood and deserves to be studied more exhaustively.

Previous studies addressing agr groups of S. aureus isolates are difficult to interpret because they were performed with samples of various sizes, encompassing dissimilar groups of isolates, i.e., methicillin-sensitive and -resistant S. aureus, from different hosts (humans, bovines), from different geographical regions, and suffering different diseases. One major advantage of our study is that it was conducted on an S. aureus population of increased homogeneity with regard to geographical region (Argentina), host involved (bovines), disease (mastitis), and antibiotic susceptibility (methicillin-sensitive S. aureus). Our results showed that a defined S. aureus lineage (agr group I, allele type A/1, mostly NT) was highly prevalent in bovines in Argentina, which lends support to the hypothesis that a few "specialized" S. aureus clones are responsible for most cases of bovine mastitis (38). This hypothesis is further supported by the study of Smith et al. (30), who described in that 87.4% of 262 bovine isolates (231 from the United States, 20 from Chile, and 11 from the United Kingdom) belonged in a single clonal complex, as defined by multilocus sequence typing. Whether the Argentine clone represented by agr group I S. aureus isolates has significant identity with the bovine clonal complex CC97 described by Smith et al. is not known and deserves further investigation. These S. aureus clones may bear a number of selected accessory elements, including individual virulence factors and pathogenicity islands, which would confer host specificity. Different hypotheses have been conceived to explain the underlying genetic basis of the evolution leading to the selection of such specialized clones (8, 17, 27, 40). The issue still remains a matter of controversy and becomes even more complicated by new insights into the plasticity of the S. aureus genome (9). Whether there are factors responsible for selective pressure leading to the emergence of noncapsulated agr group I S. aureus bacteria with increased capacities for internalization within epithelial cells remains undisclosed and merits further investigation.


   ACKNOWLEDGMENTS
 
We are grateful to Lorena Medina and Sabrina Soldavini for their expert technical assistance. We thank Ambrose L. Cheung (Darmouth Medical School, Hanover, NH) for providing S. aureus strain RN6911. We also thank the anonymous reviewers whose comments and constructive criticisms helped us to improve our manuscript.

This work was supported in part by grants from ANPCyT (PICT 08/11740 and PICT 05/10648), CONICET (PIP5933), and Universidad de Buenos Aires (UBACyT M-009), Buenos Aires, Argentina.


   FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Microbiología, Parasitología e Inmunología, Facultad de Medicina, Universidad de Buenos Aires, Paraguay 2155 p12, C1121ABG Buenos Aires, Argentina. Phone: (5411) 5950 9500, ext. 2180. Fax: (5411) 4964 2554. E-mail: sordelli{at}fmed.uba.ar. Back

{triangledown} Published ahead of print on 4 December 2006. Back

Editor: V. J. DiRita


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
1. Almeida, R. A., K. R. Matthews, E. Cifrian, A. J. Guidry, and S. P. Oliver. 1996. Staphylococcus aureus invasion of bovine mammary epithelial cells. J. Dairy Sci. 79:1021-1026.[Abstract]
2. Bannerman, T. L. 2003. Staphylococcus, Micrococcus and other catalase-positive cocci that grow aerobically, p. 384-404. In P. R. Murray, E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R. H. Yolken (ed.), Manual of clinical microbiology. American Society for Microbiology, Washington, DC.
3. Brouillette, E., and F. Malouin. 2005. The pathogenesis and control of Staphylococcus aureus-induced mastitis: study models in the mouse. Microbes Infect. 7:560-568.[CrossRef][Medline]
4. Buzzola, F. R., L. Quelle, M. I. Gomez, M. Catalano, L. Steele-Moore, D. Berg, E. Gentilini, G. Denamiel, and D. O. Sordelli. 2001. Genotypic analysis of Staphylococcus aureus from milk of dairy cows with mastitis in Argentina. Epidemiol. Infect. 126:445-452.[CrossRef][Medline]
5. Cocchiaro, J. L., M. I. Gómez, A. Risley, R. Solinga, D. O. Sordelli, and J. C. Lee. 2006. Molecular characterization of the capsule locus from non-typeable Staphylococcus aureus. Mol. Microbiol. 59:948-960.[CrossRef][Medline]
6. Dufour, P., S. Jarraud, F. Vandenesch, T. Greenland, R. P. Novick, M. Bes, J. Etienne, and G. Lina. 2002. High genetic variability of the agr locus in Staphylococcus species. J. Bacteriol. 184:1180-1186.[Abstract/Free Full Text]
7. Gilot, P., G. Lina, T. Cochard, and B. Poutrel. 2002. Analysis of the genetic variability of genes encoding the RNAIII-activating components Agr and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis. J. Clin. Microbiol. 40:4060-4067.[Abstract/Free Full Text]
8. Gilot, P., and W. van Leeuwen. 2004. Comparative analysis of agr locus diversification and overall genetic variability among bovine and human Staphylococcus aureus isolates. J. Clin. Microbiol. 42:1265-1269.[Abstract/Free Full Text]
9. Goerke, C., and C. Wolz. 2004. Regulatory and genomic plasticity of Staphylococcus aureus during persistent colonization and infection. Int. J. Med. Microbiol. 294:195-202.[CrossRef][Medline]
10. Goerke, C., S. Esser, M. Kummel, and C. Wolz. 2005. Staphylococcus aureus strain designation by agr and cap polymorphism typing and delineation of agr diversification by sequence analysis. Int. J. Med. Microbiol. 295:67-75.[CrossRef][Medline]
11. Han, H. R., S. I. Pak, S. W. Kang, W. S. Jong, and C. J. Youn. 2000. Capsular polysaccharide typing of domestic mastitis-causing Staphylococcus aureus strains and its potential exploration of bovine mastitis vaccine development. I. Capsular polysaccharide typing, isolation and purification of the strains. J. Vet. Sci. 1:53-60.[Medline]
12. Hebert, A., K. Sayasith, S. Senechal, P. Dubreuil, and J. Lagace. 2000. Demonstration of intracellular Staphylococcus aureus in bovine mastitis alveolar cells and macrophages isolated from naturally infected cow milk. FEMS Microbiol. Lett. 193:57-62.[Medline]
13. Huynh, H. T., G. Robitaille, and J. D. Turner. 1991. Establishment of bovine mammary epithelial cells (MAC-T): an in vitro model for bovine lactation. Exp. Cell Res. 197:191-199.[CrossRef][Medline]
14. Jarraud, S., G. J. Lyon, A. M. Figueiredo, L. Gerard, F. Vandenesch, J. Etienne, T. W. Muir, and R. P. Novick. 2000. Exfoliatin-producing strains define a fourth agr specificity group in Staphylococcus aureus. J. Bacteriol. 182:6517-6522.[Abstract/Free Full Text]
15. Ji, G., R. Beavis, and R. P. Novick. 1997. Bacterial interference caused by autoinducing peptide variants. Science 276:2027-2030.[Abstract/Free Full Text]
16. Lee, J. C., M. J. Liu, J. Parsonnet, and R. D. Arbeit. 1990. Expression of type 8 capsular polysaccharide and production of toxic shock syndrome toxin 1 are associated among vaginal isolates of Staphylococcus aureus. J. Clin. Microbiol. 28:2612-2615.[Abstract/Free Full Text]
17. Lindsay, J. A., C. E. Moore, N. P. Day, S. J. Peacock, A. A. Witney, R. A. Stabler, S. E. Husain, P. D. Butcher, and J. Hinds. 2006. Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes. J. Bacteriol. 188:669-679.[Abstract/Free Full Text]
18. Mayer, S. J., A. E. Waterman, P. M. Keen, N. Craven, and F. J. Bourne. 1998. Oxygen concentration in milk of healthy and mastitic cows and implications of low oxygen tension for the killing of Staphylococcus aureus by bovine neutrophils. J. Dairy Res. 55:513-519.
19. Mullarky, I. K., C. Su, N. Frieze, Y. H. Park, and L. M. Sordillo. 2001. Staphylococcus aureus agr genotypes with enterotoxin production capabilities can resist neutrophil bactericidal activity. Infect. Immun. 69:45-51.[Abstract/Free Full Text]
20. National Research Council. 1996. Guide for the care and use of laboratory animals (NIH guide, revised). National Research Council, Washington, DC.
21. Nickerson, S., W. Owens, and R. Bodie. 1995. Mastitis in dairy heifers: initial studies on prevalence and control. J. Dairy Sci. 78:1607-1618.[Abstract]
22. O'Riordan, K., and J. C. Lee. 2004. Staphylococcus capsular polysaccharide. Clin. Microbiol. Rev. 17:218-234.[Abstract/Free Full Text]
23. Pitcher, D., N. Saundres, and R. Owen. 1989. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett. Appl. Microbiol. 8:151-153.
24. Pohlmann-Dietze, P., M. Ulrich, K. B. Kiser, G. Doring, J. C. Lee, J. M. Fournier, K. Botzenhart, and C. Wolz. 2000. Adherence of Staphylococcus aureus to endothelial cells: influence of capsular polysaccharide, global regulator agr, and bacterial growth phase. Infect. Immun. 68:4865-4871.[Abstract/Free Full Text]
25. Pragman, A. A., J. M. Yarwood, T. J. Tripp, and P. M. Schlievert. 2004. Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus. Infect. Immun. 186:2430-2438.
26. Quelle, L. S., A. Corso, M. Galas, and D. O. Sordelli. 2003. STAR gene restriction profile analysis in epidemiological typing of methicillin-resistant Staphylococcus aureus: description of the new method and comparison with other polymerase chain reaction (PCR)-based methods. Diagn. Microbiol. Infect. Dis. 47:455-464.[CrossRef][Medline]
27. Robinson, D. A., A. B. Monk, J. E. Cooper, E. J. Feil, and M. C. Enright. 2005. Evolutionary genetics of the accessory gene regulator (agr) locus in Staphylococcus aureus. J. Bacteriol. 187:8312-8321.[Abstract/Free Full Text]
28. Ruegg, P. L. 2003. Investigation of mastitis problems on farms. Vet. Clin. N. Am. Food Anim. Pract. 19:47-73.[CrossRef]
29. Sandholm, M., L. Kaartinen, and S. Pyorala. 1990. Bovine mastitis—why does antibiotic therapy not always work? An overview. Vet. Pharmacol. Ther. 13:248-260.
30. Smith, E. M., L. E. Green, G. F. Medley, H. E. Bird, L. K. Fox, Y. H. Schukken, J. V. Kruze, A. J. Bradley, R. N. Zadoks, and C. G. Dowson. 2005. Multilocus sequence typing of intercontinental bovine Staphylococcus aureus isolates. J. Clin. Microbiol. 43:4737-4743.[Abstract/Free Full Text]
31. Sordelli, D. O., F. R. Buzzola, M. I. Gómez, L. Steele-Moore, D. Berg, E. Gentilini, M. Catalano, A. J. Reitz, T. Tollersrud, G. Denamiel, P. Jeric, and J. C. Lee. 2000. Capsule expression by bovine isolates of Staphylococcus aureus from Argentina: genetic and epidemiologic analyses. J. Clin. Microbiol. 38:846-850.[Abstract/Free Full Text]
32. Takeuchi, S., T. Maeda, N. Hashimoto, K. Imaizumi, T. Kaidoh, and Y. Hayakawa. 2001. Variation of the agr locus in Staphylococcus aureus isolates from cows with mastitis. Vet. Microbiol. 79:267-274.[CrossRef][Medline]
33. Tollersrud, T., K. Kenny, A. J. Reitz, and J. C. Lee. 2000. Genetic and serologic evaluation of capsule production by bovine mammary isolates of Staphylococcus aureus and other Staphylococcus spp. from Europe and the United States. J. Clin. Microbiol. 38:2998-3003.[Abstract/Free Full Text]
34. Tuchscherr, L. P. N., F. R. Buzzola, L. P. Alvarez, R. L. Caccuri, J. C. Lee, and D. O. Sordelli. 2005. Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice. Infect. Immun. 73:7932-7937.[Abstract/Free Full Text]
35. Tuchscherr, L., M. I. Gómez, D. Centrón, J. C. Lee, and D. O. Sordelli. 2002. Presence of an IS431-like element is associated with loss of the capsule locus in nontypeable Staphylococcus aureus isolates, abstr. B-186, p. 64. Abstr. 102nd Gen. Meet. Am. Soc. Microbiol. 2002. American Society for Microbiology, Washington, DC.
36. van Belkum, A., J. Kluytmans, W. van Leeuwen, R. Bax, W. Quint, E. Peters, A. Fluit, C. Vandenbroucke-Grauls, A. van den Brule, H. Koeleman, W. Melchers, J. Meis, A. Elaichouni, M. Vaneechoutte, F. Moonens, N. Maes, M. Struelens, F. Tenover, and H. Verbrugh. 1995. Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains. J. Clin. Microbiol. 33:1537-1547.[Abstract]
37. van Leeuwen, W. B., W. van Nieuwenhuizen, C. Gijzen, H. Verbrugh, and A. van Belkum. 2000. Population studies of methicillin-resistant and -sensitive Staphylococcus aureus strains reveal a lack of variability in the agrD gene, encoding a staphylococcal autoinducer peptide. J. Bacteriol. 182:5721-5729.[Abstract/Free Full Text]
38. van Leeuwen, W. B., D. C. Melles, A. Alaidan, M. Al-Ahdal, H. A. M. Boelens, S. V. Snijders, H. Wertheim, E. van Duijkeren, J. K. Peeters, P. J. van der Spek, R. Gorkink, G. Simons, H. A. Verbrugh, and A. van Belkum. 2005. Host- and tissue-specific pathogenic traits of Staphylococcus aureus. J. Bacteriol. 187:4584-4591.[Abstract/Free Full Text]
39. Wesson, C. A., L. E. Liou, K. M. Todd, G. A. Bohach, W. R. Trumble, and K. W. Bayles. 1998. Staphylococcus aureus agr and sar global regulators influence internalization and induction of apoptosis. Infect. Immun. 66:5238-5243.[Abstract/Free Full Text]
40. Wright, J. S., III, K. E. Traber, R. Corrigan, S. A. Benson, J. M. Musser, and R. P. Novick. 2005. The agr radiation: an early event in the evolution of staphylococci. J. Bacteriol. 187:5585-5594.[Abstract/Free Full Text]

Infection and Immunity, February 2007, p. 886-891, Vol. 75, No. 2
0019-9567/07/$08.00+0     doi:10.1128/IAI.01215-06
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