Skip Navigation

The Arginine Catabolic Mobile Element and Staphylococcal Chromosomal Cassette mec Linkage: Convergence of Virulence and Resistance in the USA300 Clone of Methicillin-Resistant Staphylococcus aureus

  1. Binh An Diep1,2,
  2. Gregory G. Stone3,a,
  3. Li Basuino1,
  4. Christopher J. Graber1,
  5. Alita Miller3,
  6. Shelley-Ann des Etages3,
  7. Alison Jones3,
  8. Amy M. Palazzolo-Ballance4,
  9. Françoise Perdreau-Remington1,
  10. George F. Sensabaugh2,
  11. Frank R. DeLeo4 and
  12. Henry F. Chambers1
  1. 1 Division of Infectious Diseases, Department of Medicine, San Francisco General Hospital, University of California, San Francisco
  2. 2 School of Public Health, University of California, Berkeley
  3. 3 Pfizer, Inc., Groton Laboratories, Groton, Connecticut
  4. 4 Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana
  1. Reprints or correspondence: Dr. Henry Chambers, Div. of Infectious Diseases, San Francisco General Hospital, University of California, San Francisco, Bldg. 30, Rm. 3400, 1001 Potrero Ave., San Francisco, CA 94110 (hchambers{at}medsfgh.ucsf.edu).

  1. Presented in part: 8th annual meeting of the Network on Antimicrobial Resistance in Staphylococcus aureus, Reston, Virginia, 5 March 2007.

  • ↵a Present affiliation: AstraZeneca Pharmaceuticals, Waltham, Massachusetts.

 
Next Section

Abstract

The epidemic character of community-associated methicillin-resistant Staphylococcus aureus, especially the geographically widespread clone USA300, is poorly understood. USA300 isolates carry a type IV staphylococcal chromosomal cassette mec (SCCmec) element conferringβ-lactam antibiotic class resistance and a putative pathogenicity island, arginine catabolic mobile element (ACME). Physical linkage between SCCmec and ACME suggests that selection for antibiotic resistance and for pathogenicity may be interconnected. We constructed isogenic mutants containing deletions of SCCmec and ACME in a USA300 clinical isolate to determine the role played by these elements in a rabbit model of bacteremia. We found that deletion of type IV SCCmec did not affect competitive fitness, whereas deletion of ACME significantly attenuated the pathogenicity or fitness of USA300. These data are consistent with a model in which ACME enhances growth and survival of USA300, allowing for genetic “hitchhiking” of SCCmec. SCCmec in turn protects against exposure toβ-lactams.

Factors responsible for the emergence of communityassociated methicillin-resistant Staphylococcus aureus (CA-MRSA) are poorly understood. In the absence of antibiotic pressure, the staphylococcal chromosomal cassette mec (SCCmec) element conferring β-lactam antibiotic class resistance is thought to reduce the biological fitness of MRSA [1–3]. However, strains of CA-MRSA carry the type IV allotype of SCCmec, which is smaller than those classically found in nosocomial strains. This may impose less of a fitness cost because it does not contain resistance genes other than penicillinbinding protein 2a-encoding mecA. CA-MRSA may also have other genomic adaptations to overcome the presumed fitness costs of SCCmec. Adjacent to the SCCmec element of USA300 is a putative pathogenicity island, arginine catabolic mobile element (ACME) [4]. Physical linkage between ACME and SCCmec suggests that selection for pathogenicity and selection for antibiotic resistance are interconnected. The SCCmec-ACME linkage was first discovered in the CA-MRSA USA300 genome [4]. Type I ACME in USA300 appears to have been acquired from Staphylococcus epidermidis, although the mechanism of horizontal transfer is unknown [4]. We demonstrate here that ACME can be mobilized both coordinately and independently of SCCmec via the same mechanism used for SCCmec mobilization. This allowed for generation of SCCmec and ACMEdeletion mutants in a USA300 clinical isolate to assess the role of these elements in vivo. Data are consistent with a model in which the epidemicity of USA300 is linked to type IV SCCmec, engendering no biological cost, and ACME, conferring enhanced pathogenicity or fitness.

Previous SectionNext Section

Methods

Clinical isolates of S. aureus. SF8300 is a wound isolate of pulsed-field type USA300-0114, a subtype implicated in severe disease and in numerous outbreaks. SF8300ax (ACME deletion mutant), SF8300ex (SCCmec deletion mutant), and SF8300aex (ACME-SCCmec double-deletion mutant) are isogenic derivatives of SF8300 (see below). Clinical isolates (n = 2133) used in the ACME survey contained both methicillin-susceptible S. aureus (MSSA) and MRSA that had been collected during longitudinal and population-based studies of human disease and nasal colonization conducted in the San Francisco area from 1996 through 2006 [5–8].

ACME survey. Clinical S. aureus isolates were screened for the presence of arc and opp3 gene clusters by polymerase chain reaction (PCR)—based assays, using the primer pairs AIPS.27 and AIPS.28 for arcA and AIPS.45 and AIPS.46 for opp3AB (table 1). The arc gene cluster encodes for an arginine deiminase pathway and opp3 encodes for an oligopeptide permease system, both of which are surrogate markers for type I ACME [4]. ACME-positive S. aureus isolates were further characterized by a PCR-based scanning method using 30 primer pairs (table 1), which amplified 1–2-kb segments that overlapped with adjacent segments at both ends to scan the type I ACME island found in USA300. This systematic PCR-based scanning approach had been used to identify variations in gene content, gene synteny, and other structural changes in Escherichia coli O157 [9].

Excision of ACME and/or SCCmec. pSR2, a tetracyclineselectable and temperature-sensitive plasmid containing the ccrAB2 gene complex, was electroporated into SF8300 as described by Katayama et al. [10]. SF8300(pSR2) was passaged for 3 days in tryptic soy broth (TSB) supplemented with 10 μg/mL tetracycline at 30°C. Genomic DNA was used in PCR assays to evaluate the excision of ACME, SCCmec, or ACME-SCCmec composite islands by means of primers flanking the termini of these elements. Excision of ACME was detected with the primer pair X1 and X5, SCCmec with the pair X2 and X3, and the ACME-SCCmec composite island with the pair X1 and X3. Circular extrachromosomal DNA formed on excision of these elements was assayed by PCR, using primer pairs X2 and X4 for circularized ACME, X5 and X6 for circularized SCCmec, and X4 and X6 for the circularized ACME-SCCmec composite element. PCR products were purified by means of the QIAquick PCR Purification Kit (Qiagen) and were used in DNA sequencing (University of California, Berkeley, DNA Sequencing Facility). The primers used in this study, designed from the USA300 genomic sequence (GenBank accession no. CP000255), included X1 (5'- GAATGAACGTGGATTTAATGTCC-3'), X2 (5'- CCTTCACTTAGCACTGAGG-3'), X3 (5'-TCAAACCACAATCCACAGTC- 3'), X4 (5'-GGAAATGAGCTGAAAGCACG- 3'), X5 (5'-CGTTATGGAGGTGCTCTG-3'), and X6 (5'-CTGCGGAGGCTAACTATGTC- 3').

Excision of ACME, SCCmec, and ACME-SCCmec composite islands was also screened from individual colonies of SF8300(pSR2) after 3 days of serial passage, using PCR assays as already described. Growth at the nonpermissive temperature of 42.5°C in drug-free TSB allowed for loss of pSR2 in the excision mutants. Pulsed-field gel electrophoresis with SmaI was performed as described elsewhere [5], to verify the loss of ACME, SCCmec, and ACME-SCCmec composite islands in the individual mutant strains.

Microarray experiments. Details on the microarray methods are given in appendix A, which appears only in the electronic edition of the Journal. In brief, bacterial cells were harvested from 30-mL cultures in TSB, shaking at 37°C until the mid-log or stationary phase was reached. Transcriptome analyses of SF8300 wild-type (SF8300wt) and isogenic SF8300ex, SF8300ax, and SF8300aex mutant strains were performed using Affymetrix GeneChip assays, in accordance with the manufacturer's protocol. Microarray data are posted on the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/; accession number GSE9308) and are summarized in table A1 in appendix A.

Rabbit bacteremia model. For in vivo competition experiments, bacterial strains were grown separately in TSB at 37°C with shaking for 16–18 h, harvested, and resuspended in 10% glycerol/PBS to a concentration of ∼5 × 108 cfu/mL, aliquoted into individual cryogenic vials, and immediately stored at -80°C. The 2 comparator strains were mixed at a ratio of ∼1:1 in 0.9% sodium chloride. Male New Zealand White rabbits were inoculated with 1mL containing∼2 ×7; 107 cfu, via the marginal ear vein. A 0.3–0.4-mL volume of blood was sampled daily from the ear artery for quantitative blood culture. Moribund animals and those that had lost >15% of baseline weight were euthanized, as stipulated in the protocol approved by the University of California, San Francisco, committee on animal research. Euthanized animals or those that died spontaneously were scored as such, and their lungs, spleens, and kidneys were removed and weighed. A 0.2–0.5-g sample from each tissue was processed for quantitative culture on a blood agar plate (BAP)

Statistical analysis. The number of colony-forming units in the mixed inoculum were determined by quantitative culture on a BAP. The input ratio of the parent to mutant was determined by transferring 144 cfu of the mixed inoculum on a BAP to trypticase soy agar (TSA) containing 10 μg/mL nafcillin; the SF8300 parent grew on nafcillin and was differentiated from the SF8300ex and SF8300aex mutants, which are inhibited by nafcillin. The output ratio of the parent to the mutant was determined for blood, lungs, spleen, and kidneys by transferring 144 cfu from a BAP to TSA containing 10 μg/mL nafcillin. A competition index (CI) for the 2 comparator strains was calculated using the following formula, which corrects the output ratios for variations in the input ratios: CI = log10 (output ratio/input ratio). The CI is bounded by the number of colony-forming units used to determine the ratio of the 2 comparator strains, with a lower limit of approximately -2.46 and an upper limit of approximately 2.46. For example, when all 144 cfu transferred to nafcillin-containing agar yielded a complete lack of growth or 100% growth, we would obtain incalculable ratios of 0:144 and 144:0, respectively; the least biased estimate of the ratio is obtained by adding the standard continuity correction of 0.5, resulting in 0.5:144.5 or 144.5:0.5, the log10 values of which are -2.46 and 2.46. A positive CI indicates enhanced tissue infectivity of the parent, and a negative CI indicates enhanced tissue infectivity of the mutant; a CI of 0 is the no-effect value. One-sample Student's t test was used to test for significant differences between the parent and the mutant.

Previous SectionNext Section

Results

Distribution of ACME among S. aureus clinical isolates. A longitudinal and population-based sample of 2133 S. aureusisolates was surveyed for the presence of the ACME arcand opp3 gene clusters that encode for an arginine deiminase pathway and oligopeptide permease system, respectively [4]. The arcgene cluster was detected in 60% (1271/2133) of the clinical isolates. All arc-positive isolates were clustered within 3 distinct S. aureus clonal lineages (figure 1) that were methicillin resistant, which may indicate a permissive role of the SCCmec element for acquisition of ACME. All but one of the 1248 USA300 isolates were positive for the arcand opp3gene clusters, confirming previous findings that type I ACME is integrated adjacent to the type IVa SCCmec in USA300 strains [4]. USA300 belonged to the sequence type (ST) 8 clonal lineage (figure 1), as determined by sequencing of 7 housekeeping gene fragments used in multilocus sequence typing. The ST8 lineage also contained other important CA-MRSA strains, such as the type IVa SCCmec-bearing USA500 strains, but none of these were arcpositive. Of the remaining 24 arc-positive MRSA isolates, none contained the opp3 gene cluster. Of these, 92% (22/24) of the arc-positive isolates belonged to the ST5 (USA100) clonal lineage, the predominant nosocomial MRSA strain found in the United States, and carried a type II SCCmec element. Two arc-positive isolates belonged to the ST59 (USA1000) lineage and carried type IVa SCCmec.

Figure 1.
View larger version:
Figure 1.

Independent horizontal acquisition of different arginine catabolic mobile element (ACME) allotypes in clinical Staphylococcus aureus isolates. The neighbor-joining tree was built from concatenated sequences of 7 housekeeping gene fragments used in multilocus sequence typing (SplitsTree software, version 4). Of 1248 sequence type (ST) 8 (USA300) isolates, 1247 carried the type I ACME, whereas 22 of 376 ST5 (USA100) isolates and 2 of 103 ST59 (USA1000) isolates carried a novel allotype of ACME, as determined by a polymerase chain reaction-based scanning method (table 1). The isolates from other clonal lineages that tested negative for the ACME arc cluster included the following: ST1 (USA400), n = 11; ST36 (USA200), n = 44; ST8 (USA500), n = 141; ST30 (USA1100), n = 120; and the low-frequency clonal lineages (e.g., ST12 and ST20), n = 144. The scale bar indicates evolutionary distance (substitutions per nucleotide position).

The absence of the opp3gene cluster in the arc-positive ST5 and ST59 strains indicated a potentially novel ACME allotype. To characterize this novel ACME allotype further, we used a PCR-based scanning method to amplify 1–2-kb overlapping segments covering the entire type I ACME island found in USA300. The ST5 and ST59 isolates yielded PCR products that corresponded to only the arccluster gene and flanking regions in the USA300 type I ACME (SAUSA300_0061-SAUSA300_0066; GenBank accession number NC007793). Sequencing these products yielded a 9-kb DNA contig that showed only a singlenucleotide polymorphism differentiating ST5 and ST59, strongly indicative of their common origin. The 9-kb DNA contig exhibited substantial sequence identity to USA300 through the arc gene cluster—9 and 10 substitutions for ST5 and ST59, respectively. Coupled with the observation that the PCR scan yielded no PCR amplicons for regions outside the arcgene cluster, such as the absence of the opp3 gene cluster, the data indicate that the arcgene cluster in the ST5 and ST59 isolates resides in a novel allotype of ACME distinct from the type I ACME found in USA300.

Mobilization of ACME and SCCmec by CcrAB. Because ACME is adjacent to SCCmec and is integrated into the same attBsite within orfX (figure 2), we hypothesized that mobilization and transfer of ACME could be mediated by the same recombinases (ccrAB)that are encoded by and mobilize SCCmec. Specifically, CcrAB could catalyze the site-specific recombination event between the direct repeat (DR) and inverted repeat (IR) sequences flanking each element, generating an extrachromosomal DNA circle corresponding to the excised element [10]. To test this hypothesis, we provided in trans ccrAB2via plasmid pSR2 in SF8300 and assayed for excision using PCR assays with primers flanking the termini of ACME and SCCmec, as illustrated in figure 2. Extrachromosomal DNA circles formed on excision of these elements could be detected by PCR in SF8300(pSR2) but not SF8300, using primers X2 and X4 for circularized ACME, X5 and X6 for circularized SCCmec, and X4 and X6 for circularized ACME-SCCmec elements. The chromosomal site devoid of each element could be detected by PCR in SF8300(pSR2) but not SF8300, using primers X1 and X5 for ACME excision, X2 and X3 for SCCmec excision, and X1 and X3 for the ACME-SCCmec composite island excision. DNA sequencing analysis of the PCR products not only confirmed the excision of ACME, SCCmec, or ACME-SCCmec but also provided evidence that the DR/IR sequences found at the termini of these elements were substrates for precise excision catalyzed by CcrAB (figure 2).

Figure 2.
View larger version:
Figure 2.

Precise excision of the arginine catabolic mobile element (ACME) and/or staphylococcal chromosomal cassette mec (SCCmec) mediated by ccrA/B. The arrows indicate the locations of primers used to characterize the ACME and/or SCCmec excision products. For example, polymerase chain reaction products for the primer pair X1 and X5 and the primer pair X2 and X4 indicate, respectively, the excision of ACME from the chromosome and the formation of the extrachromosomal circularized ACME. DR, direct repeat; IR, inverted repeat.

The circularized ACME contained a novel attACME attachment sequence, which was presumably formed by head-to-ligation of DRACMEatt-L and IRACME-L sequences (figure 2); attACME differed from attSCCmec and attACME-SCCmec sequences in its lack of a right IR sequence (e.g., IRSCC-R). It remains to be determined whether the novel attACME could serve as a substrate for ccrAB2-mediated integration or whether ACME is cotransferred with an associated SCC element using the canonical attSCCmec (or attACME-SCCmec) sequence.

Because CcrAB2 excised these elements from the chromosome at high frequency, we were able to establish the 3 SF8300 mutants: (1) the SCCmec deletion mutant, SF8300ex; (2) the ACME deletion mutant, SF8300ax; and (3) the ACME-SCCmec double-deletion mutant, SF8300aex. Pulsed-field gel electrophoresis of SmaI digests of chromosomal DNA confirmed the excision of these elements in the mutant strains, resulting in the loss of ∼24 kb in SF8300ex, 31 kb in SF8300ax, and 55kb in SF8300aex, compared with the corresponding fragment of the SF8300 parental strain (figure 3). There were no differences in in vitro growth rates between SF8300, SF8300ex, SF8300ax, and SF8300aex strains (data not shown).

Figure 3.
View larger version:
Figure 3.

Pulsed-field gel electrophoresis of SmaI digested chromosomal DNA of SF8300 (lane 1); the staphylococcal chromosomal cassette mec (SCCmec) excision mutant, SF8300ex (lane 2); the arginine catabolic mobile element (ACME) excision mutant, SF8300ax (lane 3); and the ACME-SCCmec excision mutant, SF8300aex (lane 4).

Enhancement of competitive fitness by ACME but not SCCmec in a rabbit bacteremia model. Because there are no suspected virulence determinants within the SCCmec element, we hypothesized that elimination of SCCmec would have no effect on the virulence of SF8300. On the other hand, elimination of type I ACME in SF8300 could result in a competitive fitness cost because this putative pathogenicity island contains several candidate virulence determinants [4]. To test these hypotheses, we first conducted competition experiments in a rabbit bacteremia model in which 18 rabbits were coinfected with SF8300 and the SCCmec deletion mutant, SF8300ex, at a ratio of ∼1:1. The bacterial burden in organs was determined in rabbits that succumbed to infection or moribund rabbits that were euthanized. The total number of colony-forming units in each organ was expressed as the log10 value of the colony-forming units per organ, except for blood, for which the result was expressed as the log10 value of the colony-forming units per milliliter (table 2). The output ratios of the SF8300 parent to SF8300ex mutant in tissues were corrected for variations in the input ratios of the inoculum and represent the CI. The CIs for lungs, spleen, kidneys, and blood did not differ significantly from 0 (the no-effect value), indicating that elimination of SCCmec had no effect on virulence (table 2 and figure 4A). Therefore, methicillin resistance has no direct role in MRSA pathogenesis.

Figure 4.
View larger version:
Figure 4.

The arginine catabolic mobile element (ACME) but not staphylococcal chromosomal cassette mec (SCCmec) contributes to virulence in a rabbit bacteremia model. SF8300 and SF8300ex (SCCmec deletion mutant) (A) or SF8300 and SF8300aex (ACME-SCCmec deletion mutant) (B) were coinoculated via the marginal ear vein of New Zealand White rabbits. Each geometric symbol represents the competition index, calculated as log10 (output ratio/input ratio)—that is, the logarithm of the output ratios of the parent and isogenic mutant in lungs, spleen, kidneys, or blood of a single animal after correction for variations in input ratios. The geometric means are indicated by horizontal bars. The competition index is bounded by the detection limits of the experimental assay, which ranged from -2.46 to 2.46. The null hypothesis (competition index of 0) that there was no difference in competitive fitness between the parent and isogenic mutant was tested using a 1-sample Student's t test. *P < .05 (2-tailed).

Because SCCmec did not attenuate fitness, it provided a convenient marker for evaluating the role played by ACME in the rabbit bacteremia model. For this experiment, 16 rabbits were coinfected with SF8300 and the ACME-SCCmec doubleexcision mutant, SF8300aex. Importantly, the SF8300 parental strain exhibited significantly enhanced fitness compared with SF8300aex, with a mean CI of 0.91 for blood, 0.88 for the lungs, and 0.55 for the spleen (figure 4B and table 2). Because elimination of SCCmec had no effect on competitive fitness, the attenuated fitness in SF8300aex can be attributed to the deletion of ACME. Of note, infection with the SF8300-SF8300ex pair was associated with significantly more rapidly fatal disease than infection with the SF8300-SF8300aex pair (P < .001), despite a lower bacterial inoculum used in the SF8300-SF8300ex experiment (table 2). Because the SF8300-SF8300ex and SF8300-SF8300aex experiments were conducted at different times, the observed differences in mean survival time and inoculum size could be due to interexperiment variability. However, these data lead to a hypothesis that elimination of ACME significantly reduces bacterial infectivity of target organs, which could be correlated with less rapidly fatal disease.

Table 1.
View larger version:
Table 1.

Primers used in polymerase chain reaction scanning of type I arginine catabolic mobile element.

Table 2.
View larger version:
Table 2.

Experimental conditions, survival, and bacterial burdens in target organs of rabbits coinfected with either SF8300 and SF8300ex or SF8300 and SF8300aex.

Table A1.
View larger version:
Table A1.

Analysis of the deletion of the arginine catabolic mobile element (ACME) and/or staphylococcal chromosomal cassette mec (SCCmec) in SF8300 at the stationary phase of growth.

Table A2.
View larger version:
Table A2.

Analysis of the deletion of the arginine catabolic mobile element (ACME) and/or staphylococcal chromosomal cassette mec (SCCmec) in SF8300 at the exponential phase of growth.

No effect of the ACME and SCCmec deletions on global gene expression. To determine whether attenuated virulence with deletion of ACME could be due to an effect on global gene expression, we compared the transcriptomes of SF8300ex, SF8300ax, and SF8300aex with SF8300 by means of an expression microarray that contained∼95% of identified open reading frames of the sequenced USA300-0114. As expected, many genes contained within SCCmec (SAUSA300_0027-SAUSA300_0044) and ACME (SAUSA300_0047-SAUSA300_0079) were expressed in SF8300 but not in SF8300ex, SF8300ax, or SF8300aex (tables A1 and A2 in appendix A). Notably, at the stationary phase of growth, there were no significant changes in transcript levels among wild-type and isogenic mutant strains beyond those encoded by SCCmec and ACME (table A1). At the exponential phase of growth, 19 non-SCCmec transcripts in SF8300ex and 17 non-ACME transcripts in SF8300ax met the criteria for differentially regulated genes (table A2). Of these, only SAUSA300_0216 (encoding a hexose phosphate transporter) and SAUSA300_1193 (encoding glycerol-3-phosphate dehydrogenase) were also differentially expressed in SF8300aex, indicating that deletion of SCCmec and ACME did not alter global gene expression in USA300. Taken together, these findings suggest that attenuated virulence of SF8300aex (figure 4B) is due to ACME and not some pleiotropic alterations resulting in the deletions of ACME and/or SCCmec.

Previous SectionNext Section

Discussion

The overall objective of our study was to gain new insight into the molecular basis of the worldwide emergence of CA-MRSA. We demonstrated that elimination of SCCmec had no effect on competitive fitness, whereas elimination of ACME significantly reduced fitness in a rabbit model (table 2 and figure 4). Three important inferences can be drawn from these results. First, CA-MRSA could emerge and persist in communities because the type IV SCCmec imposes no fitness cost either in vitro and in vivo (the rabbit model). Mathematical models predict that low levels of antibiotic use in communities are sufficient to drive the spread of resistant bacteria when antibiotic resistance engenders no biological cost [11, 12]. Accordingly, acquisition of the type IV SCCmec by genetically diverse MSSA has given rise to stable populations of CA-MRSA strains in communities worldwide [13, 14]. In contrast, the burden other SCCmec allotypes (types I, II, and III) impose on the bacterial fitness (i.e., decreased in vitro growth rate [2, 3]) has probably restricted classic nosocomial strains bearing these elements from spreading into the community; hospital-associated MRSA seems to critically depend on high rates of antibiotic use in hospitals to overcome the fitness burden of antibiotic resistance.

Second, because elimination of SCCmec engenders no fitness cost, the fitness cost associated with elimination of both SCCmec and ACME in USA300 can be attributed to deletion of ACME (table 2 and figure 4). Type I ACME exhibits many characteristics that conform to the pathogenicity island concept [15]. One is that it enhances hematogenous dissemination to target organs. Another is that it is found in one of the most pathogenic strains of S. aureus tested in animal models [16, 17] while being rarely present in other S. aureus strains (figure 1), is present in 19% of S. epidermidisstrains (a major cause of device-related nosocomial infections), and is absent in nonpathogenic Staphylococcus species (B.A.D., C.J.G., and H.F.C., unpublished data). Yet another is that ACME contains 2 gene clusters-arc, encoding the arginine deiminase pathway, and opp3, encoding an oligopeptide permease system [4]-that are homologues of genetic elements that have been implicated in pathogenesis [18–20]. Finally, ACME is adjacent to SCCmec and mobilized by the same ccrAB that mobilizes SCCmec. Taken together, it seems that horizontal acquisition of type I ACME has transformed a single precursor cell to USA300 and conferred pathogenicity or fitness to this epidemic-prone clone.

Third, a molecular genetic basis for the epidemicity of USA300 [8, 21–35] could be explained by the interconnection between selection for antibiotic resistance and selection for pathogenicity. In longitudinal and population-based analyses of clinical S. aureus isolates, we discovered not only that USA300 accounted for 59% of all S. aureus infections but also that nearly all (1247/1248) USA300 isolates carried type I ACME and type IV SCCmec. In community settings largely devoid of antibiotic selection pressure, ACME could enhance growth, survival, and dissemination of USA300, thus allowing for the genetic “hitchhiking” of SCCmec. SCCmec protects against exposure to β-lactams, further enhancing rapid dissemination of USA300 with antibiotic use [21, 34, 35]. SCCmec also has another essential functionality in that it provides the CcrAB recombinases that could mediate mobilization and integration of ACME into the chromosome (figure 2) [10].

USA300 is remarkable for its capacity to displace other CA-MRSA strains containing type IV SCCmec, including ST1 (USA400), ST30 (USA1100), and ST80 [25, 36, 37]. It is of interest that these CA-MRSA strains all carry a 2-component poreforming toxin, Panton-Valentine leukocidin (PVL) [8, 38]. It is not possible on the basis of epidemiological data alone to establish whether PVL directly contributes to CA-MRSA pathogenesis [39]. Discordant results from mouse infection models, however, suggest either that PVL has no effect on host-pathogen interaction [17] or that its activity could be limited to the lungs at high doses [40]. Attributing CA-MRSA pathogenicity to PVL alone ignores the possible contributions of other virulence determinants unique to USA300, including the pyrogenic toxin superantigens (SEK and SEQ) and other exoproteins (SCN, SAK, and CHP) that could allow USA300 to evade and subvert host defenses [4]. Taken together, the data are consistent with a model in which type IACMEconfers the striking epidemic character of USA300 but type IV SCCmec and/or PVL do not, because the former is found only in USA300 and the latter are associated with the vast majority of CA-MRSA strains recovered worldwide [38]. Although a novel allotype of ACME was acquired independently by ST5 (USA100) and ST59 (USA1000) strains,ACMEfound in ST5 and ST59 differ significantly in gene content from the type I ACME found in USA300, including the lack of the opp3 operon that could contribute to virulence. It remains to be determined whether this novel allotype of ACME also confers pathogenicity or fitness to ST5 and ST59 strains.

Previous SectionNext Section

Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: US Public Health Service (grant R01/CCR923381 to H.F.C.); Microbial Pathogenesis and Host Defense postdoctoral fellowship from the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH) (5T32AI060537-02 to B.A.D.); Intramural Research Program of the NIAID, NIH (support to F.R.D. and A.M.P.-B.)

  • Received July 26, 2007.
  • Accepted December 17, 2007.
Previous SectionNext Section

Appendix: Proofs

Previous SectionNext Section

APPENDIX A

Cells were harvested from 30-mL cultures in tryptic soy broth, with shaking at 37°C until the mid-log or stationary phase was reached. RNA isolation was performed using the Bio101 Kit (Qbiogene) in accordance with supplied protocol, except that TRIzol reagent was used instead of RNApro solution. Fragmented and biotin-dUTP—labeled cDNA was generated from purified RNA using the prokaryotic sample and array processing protocol (Affymetrix). The resulting cDNA was fragmented by use of DNase I, labeled with terminal transferase, and biotinylated at the 3' end using GeneChip DNA labeling reagent. Hybridization master mix was added to each sample, and the resultant mixtures were hybridized to Affymetrix S. aureus genome microarrays for 16 h at 45°C. After hybridization, washing and staining was done on a GeneChip Fluidics Station 450s (Affymetrix), using the ProkGE-WS2v3_450 procedure. After washing and staining, the microarrays were scanned on a GeneChip Scanner 3000.

GeneChip Operating Software (version 1.4; http://www.affymetrix.com) was used to perform the preliminary analysis of custom chips at the probe-set level. Microarray analysis was performed as described elsewhere [21], but with modifications. Data were imported into GeneSpring GX software (version 7.3; Agilent), and the similarity of biological replicates was determined by means of hierarchical clustering using a Pearson correlation similarity measure with average linkage. A principle components analysis plot was generated (Partek) as a secondary check on the similarity of biological replicates. Microarray data were combined into a custom worksheet (Microsoft Excel 2003; Microsoft), which was used to correlate replicates of all test conditions and controls. The ratios of test (wild-type strain) to control (mutant strain) were reported, along with the associated probability values from Student's t tests. P values obtained from analysis of variance (Partek) were filtered using the false-discovery rate. Gene lists were generated, with emphasis on quality, statistical filters, and ⩾2-fold change in expression. Microarray data are posted on the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/; accession number GSE9308).

Previous Section
 

References

  1. 1.↵
    1. Ito T,
    2. Okuma K,
    3. Ma XX,
    4. Yuzawa H,
    5. Hiramatsu K
    . Insights on antibiotic resistance of Staphylococcus aureus from its whole genome: genomic island SCC. Drug Resist Updat 2003;6:41-52.
    CrossRefMedlineWeb of Science
  2. 2.↵
    1. Ender M,
    2. McCallum N,
    3. Adhikari R,
    4. Berger-Bachi B
    . Fitness cost of SCCmec and methicillin resistance levels in Staphylococcus aureus. Antimicrob Agents Chemother. N Engl J Med 2004;48:2295-7.
  3. 3.↵
    1. Lee SM,
    2. Ender M,
    3. Adhikari R,
    4. Smith JM,
    5. Berger-Bachi B,
    6. Cook GM
    . Fitness cost of SCCmec and methicillin resistance levels in Staphylococcus aureus. Antimicrob Agents Chemother. N Engl J Med 2007;51:1497-9.
  4. 4.↵
    1. Diep BA,
    2. Gill SR,
    3. Chang RF,
    4. et al
    . Complete genome sequence of USA300, an epidemic clone of community-acquired meticillin-resistant Staphylococcus aureus. Lancet 2006;367:731-9.
    CrossRefMedlineWeb of Science
  5. 5.↵
    1. Charlebois ED,
    2. Bangsberg DR,
    3. Moss NJ,
    4. et al
    . Population-based community prevalence of methicillin-resistant Staphylococcus aureus in the urban poor of San Francisco. Clin Infect Dis 2002;34:425-33.
    Abstract/FREE Full Text
  6. 6.
    1. Diep BA,
    2. Sensabaugh GF,
    3. Somboona NS,
    4. Carleton HA,
    5. Perdreau-Remington F
    . Widespread skin and soft-tissue infections due to two methicillin-resistant Staphylococcus aureus strains harboring the genes for Panton-Valentine leukocidin. J Clin Microbiol 2004;42:2080-4.
    Abstract/FREE Full Text
  7. 7.
    1. Pan ES,
    2. Diep BA,
    3. Charlebois ED,
    4. et al
    . Population dynamics of nasal strains of methicillin-resistant Staphylococcus aureus—and their relation community-associated disease activity. J Infect Dis 2005;192:811-8.
    Abstract/FREE Full Text
  8. 8.↵
    1. Diep BA,
    2. Carleton HA,
    3. Chang RF,
    4. Sensabaugh GF,
    5. Perdreau-Remington F
    . Roles of 34 virulence genes in the evolution of hospital- and community-associated strains of methicillin-resistant Staphylococcus aureus. J Infect Dis 2006;193:1495-503.
    Abstract/FREE Full Text
  9. 9.↵
    1. Ohnishi M,
    2. Terajima J,
    3. Kurokawa K,
    4. et al
    . Genomic diversity of enterohemorrhagic Escherichia coli O157 revealed by whole genome PCR scanning. Proc Natl Acad Sci USA 2002;99:17043-8.
    Abstract/FREE Full Text
  10. 10.↵
    1. Katayama Y,
    2. Ito T,
    3. Hiramatsu K
    . A new class of genetic element, staphylococcus cassette chromosome mec, encodes methicillin resistance in Staphylococcus aureus. Antimicrob Agents Chemother 2000;44:1549-55.
    Abstract/FREE Full Text
  11. 11.↵
    1. Levin BR,
    2. Lipsitch M,
    3. Perrot V
    . The population genetics of antibiotic resistance. Clin Infect Dis 1997;24(Suppl 1):S9-16.
  12. 12.↵
    1. Levin BR
    . Minimizing potential resistance: a population dynamics view. Clin Infect Dis 2001;33(Suppl 3):S161-9.
  13. 13.↵
    1. Enright MC,
    2. Robinson DA,
    3. Randle G,
    4. Feil EJ,
    5. Grundmann H,
    6. Spratt BG
    . The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). Proc Natl Acad Sci USA 2002;99:7687-92.
    Abstract/FREE Full Text
  14. 14.↵
    1. Robinson DA,
    2. Enright MC
    . Evolutionary models of the emergence of methicillin-resistant Staphylococcus aureus. Antimicrob Agents Chemother 2003;47:3926-34.
    Abstract/FREE Full Text
  15. 15.↵
    1. Hacker J,
    2. Blum-Oehler G,
    3. Muhldorfer I,
    4. Tschape H
    . Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol 1997;23:1089-97.
    CrossRefMedlineWeb of Science
  16. 16.↵
    1. Voyich JM,
    2. Braughton KR,
    3. Sturdevant DE,
    4. et al
    . Insights into mechanisms used by Staphylococcus aureus to avoid destruction by human neutrophils. J Immunol 2005;175:3907-19.
    Abstract/FREE Full Text
  17. 17.↵
    1. Voyich JM,
    2. Otto M,
    3. Mathema B,
    4. et al
    . Is Panton-Valentine leukocidin the major virulence determinant in community-associated methicillinresistant Staphylococcus aureus disease? J Infect Dis 2006;194:1761-70.
    Abstract/FREE Full Text
  18. 18.↵
    1. Coulter SN,
    2. Schwan WR,
    3. Ng EY,
    4. et al
    . Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments. Mol Microbiol 1998;30:393-404.
    CrossRefMedlineWeb of Science
  19. 19.
    1. Degnan BA,
    2. Fontaine MC,
    3. Doebereiner AH,
    4. et al
    . Characterization of an isogenic mutant of Streptococcus pyogenes Manfredo lacking the ability to make streptococcal acid glycoprotein. Infect Immun 2000;68:2441-8.
    Abstract/FREE Full Text
  20. 20.↵
    1. Podbielski A,
    2. Pohl B,
    3. Woischnik M,
    4. et al
    . Molecular characterization of group A streptococcal (GAS) oligopeptide permease (opp) and its effect on cysteine protease production. Mol Microbiol 1996;21:1087-99.
    CrossRefMedlineWeb of Science
  21. 21.↵
    1. Moran GJ,
    2. Krishnadasan A,
    3. Gorwitz RJ,
    4. et al
    . Methicillin-resistant S.aureus infections among patients in the emergency department. N Engl J Med 2006;355:666-74.
    CrossRefMedline
  22. 22.
    1. King MD,
    2. Humphrey BJ,
    3. Wang YF,
    4. Kourbatova EV,
    5. Ray SM,
    6. Blumberg HM
    . Emergence of community-acquired methicillin-resistant Staphylococcus aureus USA 300 clone as the predominant cause of skin and soft-tissue infections. Ann Intern Med 2006;144:309-17.
    Abstract/FREE Full Text
  23. 23.
    1. Hota B,
    2. Ellenbogen C,
    3. Hayden MK,
    4. Aroutcheva A,
    5. Rice TW,
    6. Weinstein RA
    . Community-associated methicillin-resistant Staphylococcus aureus skin and soft tissue infections at a public hospital: do public housing and incarceration amplify transmission? Arch Intern Med 2007;167:1026-33.
    Abstract/FREE Full Text
  24. 24.
    1. Gilbert M,
    2. MacDonald J,
    3. Gregson D,
    4. et al
    . Outbreak in Alberta of community-acquired (USA300) methicillin-resistant Staphylococcus aureus in people with a history of drug use, homelessness or incarceration. CMAJ 2006;175:149-54.
    Abstract/FREE Full Text
  25. 25.↵
    1. Larsen A,
    2. Stegger M,
    3. Goering R,
    4. Sorum M,
    5. Skov R
    . Emergence and dissemination of the methicillin resistant Staphylococcus aureus USA300 clone in Denmark (2000–2005). Euro Surveill. Vol. 12. 2007.
  26. 26.
    1. Witte W,
    2. Cuny C,
    3. Strommenger B,
    4. Nübel U
    . Community MRSA ST8 (“USA300”) has arrived in Central Europe [abstract]; Program and abstracts of the 17th European Congress of Clinical Microbiology and Infectious Diseases; 2007; Munich. European Society of Clinical Microbiology and Infectious Diseases.
  27. 27.
    1. Kearns AM,
    2. Ganner M,
    3. Hill RLR,
    4. et al
    . Community-associated MRSA ST8-SCCmecIVa (USA-300): experience in England and Wales [abstract]; Program and abstracts of the 17th European Congress of Clinical Microbiology and Infectious Diseases; 2007; Munich. European Society of Clinical Microbiology and Infectious Diseases.
  28. 28.
    1. Gonzalez BE,
    2. Martinez-Aguilar G,
    3. Hulten KG,
    4. et al
    . Severe staphylococcal sepsis in adolescents in the era of community-acquired methicillinresistant Staphylococcus aureus. Pediatrics 2005;115:642-48.
    Abstract/FREE Full Text
  29. 29.
    1. Seybold U,
    2. Kourbatova EV,
    3. Johnson JG,
    4. et al
    . Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care-associated blood stream infections. Clin Infect Dis 2006;42:647-56.
    Abstract/FREE Full Text
  30. 30.
    1. Haque NZ,
    2. Davis SL,
    3. Manierski CL,
    4. et al
    . Infective endocarditis caused by USA300 methicillin-resistant Staphylococcus aureus (MRSA). Int J Antimicrob Agents 2007;30:72-7.
    CrossRefMedlineWeb of Science
  31. 31.
    1. Miller LG,
    2. Perdreau-Remington F,
    3. Rieg G,
    4. et al
    . Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles. N Engl J Med 2005;352:1445-53.
    CrossRefMedlineWeb of Science
  32. 32.
    1. Francis JS,
    2. Doherty MC,
    3. Lopatin U,
    4. et al
    . Severe community-onset pneumonia in healthy adults caused by methicillin-resistant Staphylococcus aureus carrying the Panton-Valentine leukocidin genes. Clin Infect Dis 2005;40:100-7.
    Abstract/FREE Full Text
  33. 33.
    1. Hageman JC,
    2. Uyeki TM,
    3. Francis JS,
    4. et al
    . Severe community-acquired pneumonia due to Staphylococcus aureus, 2003–04 influenza season. Emerg Infect Dis 2006;12:894-9.
    MedlineWeb of Science
  34. 34.↵
    1. Kazakova SV,
    2. Hageman JC,
    3. Matava M,
    4. et al
    . A clone of methicillinresistant Staphylococcus aureus among professional football players. N Engl J Med 2005;352:468-75.
    CrossRefMedlineWeb of Science
  35. 35.↵
    1. Hidron AI,
    2. Kourbatova EV,
    3. Halvosa JS,
    4. et al
    . Risk factors for colonization with methicillin-resistant Staphylococcus aureus (MRSA) in patients admitted to an urban hospital: emergence of community-associated MRSA nasal carriage. Clin Infect Dis 2005;41:159-66.
    Abstract/FREE Full Text
  36. 36.↵
    1. Carleton HA,
    2. Diep BA,
    3. Charlebois ED,
    4. Sensabaugh GF,
    5. Perdreau-Remington F,
    6. et al
    . Community-adapted methicillin-resistant Staphylococcus aureus (MRSA): population dynamics of an expanding community reservoir of MRSA. J Infect Dis 2004;190:1730-8.
    Abstract/FREE Full Text
  37. 37.↵
    1. Stemper ME,
    2. Brady JM,
    3. Qutaishat SS,
    4. et al
    . Shift in Staphylococcus aureus clone linked to an infected tattoo. Emerg Infect Dis 2006;12:1444-6.
    MedlineWeb of Science
  38. 38.↵
    1. Vandenesch F,
    2. Naimi T,
    3. Enright MC,
    4. et al
    . Community-acquired methicillin-resistant Staphylococcus aureus carrying Panton-Valentine leukocidin genes: worldwide emergence. Emerg Infect Dis 2003;9:978-84.
    MedlineWeb of Science
  39. 39.↵
    1. Chambers HF
    . Community-associated MRSA: resistance and virulence converge. N Engl J Med 2005;352:1485-7.
    CrossRefMedlineWeb of Science
  40. 40.↵
    1. Labandeira-Rey M,
    2. Couzon F,
    3. Boisset S,
    4. et al
    . Staphylococcus aureus Panton-Valentine leukocidin causes necrotizing pneumonia. Science 2007;315:1130-3.
    Abstract/FREE Full Text
| Table of Contents

This Article

  1. J Infect Dis. (2008) 197 (11): 1523-1530. doi: 10.1086/587907
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most