clin infect dis. 2007 appelbaum s165 70

Staphyloccocal Resistance: Microbiology • CID 2007:45 (Suppl 3) • S165

S U P P L E M E N T A R T I C L E

Microbiology of Antibiotic Resistancein Staphylococcus aureus

Peter C. AppelbaumDivision of Clinical Pathology, Penn State College of Medicine, and Clinical Microbiology, Penn State Milton S. Hershey Medical Center, Hershey,Pennsylvania

Methicillin-resistant Staphylococcus aureus (MRSA) isolates came into existence soon after the introduction

of methicillin. Historically, MRSA isolates have been associated with nosocomial infections and rapidly de-

veloped resistance to multiple drug classes. However, in recent years, different strains with unique phenotypes

have emerged in the community, and the reservoir of community-associated MRSA is rapidly expanding.

Community-associated pathogens are likely to cause life-threatening systemic infections, especially in children

and elderly individuals, and may also cause serious skin and soft-tissue infections in healthy individuals.

Compared with nosocomial strains, community-associated MRSA isolates are associated with increased vir-

ulence and currently are more likely to be susceptible to a variety of antibiotics. The epidemiological and

microbiological differences between community-associated and nosocomial MRSA infections necessitate dif-

ferent strategies to prevent and treat the 2 types of infections. Vancomycin nonsusceptibility in S. aureus is

on the increase, further complicating therapy.

Methicillin-resistant Staphylococcus aureus (MRSA) is a

major pathogen worldwide; MRSA infections are as-

sociated with increased morbidity and mortality, in

comparison with other S. aureus infections. Over the

past decade, the changing pattern of resistance in S.

aureus has underscored the need for new antimicrobial

agents [1]. Once confined to health care–associated en-

vironments, MRSA has now migrated into the com-

munity. Community-associated strains share some

characteristics with nosocomial strains but also differ

in antimicrobial susceptibility and potential virulence.

Of concern is the probable increasing prevalence of

heterogeneous vancomycin-intermediate S. aureus

(hVISA) and vancomycin-intermediate S. aureus (VISA)

MRSA strains in Europe, Asia, and the United States [1].

Although 7 cases of infection with vancomycin-resistant

S. aureus (VRSA) strains have been described in the

United States, the clinical and epidemiological signifi-

Reprints or correspondence: Dr. Peter C. Appelbaum, Dept. of Pathology, PennState Hershey Medical Center, 500 University Dr., Hershey, PA 17033([email protected]).

Clinical Infectious Diseases 2007; 45:S165–70� 2007 by the Infectious Diseases Society of America. All rights reserved.1058-4838/2007/4506S3-0002$15.00DOI: 10.1086/519474

cance of this resistance phenotype is unclear at the pres-

ent time [1].

AN OVERVIEW OF THE DEVELOPMENTOF S. AUREUS RESISTANCE

In the 1940s, penicillin was introduced for the treat-

ment of infection; as early as 1942, strains of S. aureus

resistant to penicillin had been detected in hospitals.

Within 2 decades, ∼80% of both hospital- and com-

munity-acquired S. aureus isolates were penicillin re-

sistant. The introduction of methicillin in 1961 was

rapidly followed by reports of methicillin resistance in

S. aureus. Today, MRSA strains are found worldwide,

and most are multidrug resistant [1].

Study of early isolates of MRSA showed that a key

genetic component responsible for resistance, mecA, is

not native to the S. aureus genome. The staphylococcal

chromosome cassette mec (SCCmec) has been charac-

terized as a novel, mobile resistance element that differs

from both transposons and bacteriophages [2]. MRSA

typically spreads through clones; however, it is known

that the mec gene has been transmitted between S. au-

reus strains and, possibly, between other staphylococcal

species [2].

Until the 1990s, MRSA was a pathogen associated

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S166 • CID 2007:45 (Suppl 3) • Appelbaum

with nosocomial infections. However, in the late 1990s, MRSA

began to be detected in infections that arose outside of the

health care setting. Some cases in the United States have oc-

curred in patients who have never been hospitalized and who

have no risk factors for MRSA infection [3]. In 2005, Kazakova

et al. [4] reported a community-associated MRSA (CA-MRSA)

clone isolated from US football players with skin abscesses. The

strain was susceptible to most antimicrobial agents except b-

lactams and macrolides. It carried a subtype of the SCCmec

unit (SCCmec type IV) that differed from that of health care–

associated MRSA (HA-MRSA) and a gene for Panton-Valentine

leukocidin (PVL), which may be associated with severe nec-

rotizing infections. Evaluation of the strain involved showed

that it was identical to epidemiologically unrelated CA-MRSA

isolates from various other regions of the country [4]. Simi-

larities in the genetic backgrounds of early and contemporary

epidemic clones of MRSA have also been reported [5]. Recent

work has confirmed that these differences between CA-MRSA

and HA-MRSA are typical: CA-MRSA strains usually have dif-

ferent subtypes of SCCmec (IV and V), are often resistant to

fewer antibiotic classes (frequently only b-lactams and mac-

rolides), and are more virulent, and a high proportion carry

the genes encoding PVL [3]. It is noteworthy that the impor-

tance of PVL as a determinant of virulence in CA-MRSA has

recently been called into question [6]. Although the emergence

and virulence of CA-MRSA have been associated with strains

USA300 and USA400, both containing PVL, a direct role for

PVL in increased virulence had not been systematically eval-

uated before the study by Voyich et al. [6]. A comparison of

the virulence of PVL-positive and PVL-negative strains of CA-

MRSA in mouse models has shown them to be equally virulent.

Isogenic PVL-negative (knockout) strains of USA300 and

USA400 were also found to be as virulent as the wild-type

strains [6].

Carleton et al. [7] evaluated MRSA reservoirs in the com-

munity, in an effort to understand the population dynamics.

Random sampling ( ) from 2154 MRSA isolates col-n p 490

lected during a 7-year period from both inpatients and out-

patients in San Francisco revealed that a 14-fold increase in

the frequency of methicillin resistance between 1998 and 2002

was due to 4 specific genotypes. Contrary to what might have

been postulated, these genotypes were associated primarily with

community-onset disease and were significantly less likely to

be associated with hospital- or health care–associated infections.

Thus, the reservoir of CA-MRSA seems to be rapidly increasing.

In recent literature, there are increasing reports of the emer-

gence of health care–associated infections caused by MRSA

strains with molecular characteristics of CA-MRSA, suggesting

that the phenotype of CA-MRSA may become that of HA-

MRSA in the future [8], with serious implications for therapy

for these infections.

MRSA has been associated with a variety of infections, rang-

ing from superficial to more deep rooted. Life-threatening CA-

MRSA infections have been reported, including neonatal sepsis

and community-acquired pneumonia [3, 9]. In infants treated

since birth in a neonatal intensive-care unit (NICU), 8 of 17

cases of S. aureus bacteremia were found to be due to MRSA.

Six of the MRSA strains had SCCmec genes found in com-

munity isolates; all of these were resistant to b-lactam antibi-

otics and erythromycin, and 1 was also resistant to clindamycin

[3]. A majority of infants (88%) presented with septic shock,

and, despite rapid treatment with vancomycin, 3 of the infants

died, and another 3 had long, difficult hospital courses [3].

The authors indicated that the “introduction of community

strains of MRSA into our NICU reflects the high prevalence

of these strains in the Houston community” [3, p. 1464]. They

also noted that, despite causing superficial infections in healthy

adults, CA-MRSA can be highly virulent in some hosts, such

as the young children in their study [3]. Francis et al. [9]

reported CA-MRSA infection in 4 patients with severe nec-

rotizing MRSA pneumonia who did not have typical risk factors

for MRSA infection. All were infected with strains that carried

the PVL gene and the SCCmec type IV cassette, which is typical

of community strains of MRSA. Two patients had coinfections

with influenza A virus, which the authors felt contributed to

the severity of disease. One patient died, and the other 3 re-

quired prolonged hospitalization as a result of significant com-

plications [9].

KEY FEATURES OF MRSA

Methicillin resistance in S. aureus involves an altered target site

due to an acquired penicillin-binding protein (PBP 2a) with

decreased affinity to b-lactams [2]. The mecA gene encodes this

protein and is located on a mobile SCCmec cassette chromo-

some [10, 11]. This genetic element confers resistance to most

currently available b-lactam antibiotics [2]. However, not all

mecA clones are resistant to methicillin, and overall resistance

levels in a population of MRSA depend on efficient production

of PBP 2a, which is modulated by a variety of chromosomal

factors. This explains why MRSA resistance levels range from

phenotypically susceptible to highly resistant [2].

There are 5 SCCmec subtypes (types I–V), which vary in size

from ∼20 kb to 68 kb. The smaller subtypes (I, IV, and V)

encode only recombinase genes and the structural and regu-

latory genes for resistance to methicillin; they do not carry

transposable elements and genes encoding resistance to non–

b-lactam antibiotics [12]. HA-MRSA isolates typically have

SCCmec subtypes I–III and rarely carry the gene for PVL. In

contrast, types IV and V are associated with CA-MRSA isolates,



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and at least type IV frequently carries PVL [12]. Healy et al.

[3, p. 1465] note that the SCCmec cassette in community strains

is smaller than that in HA-MRSA and speculate that “its genetic

composition likely enhances mobility and ability to be trans-

ferred between strains.”

Methicillin resistance in S. aureus is expressed in a stepwise

fashion [11]. In the pre-MRSA that has the mecA gene together

with regulator genes mecI and mecR1, S. aureus strains do not

express methicillin resistance. In the hetero-MRSA, the mecI-

mediated repression is released by mutation, and the strain

becomes resistant to low concentrations of methicillin but re-

mains susceptible to high concentrations. Finally, a homo-

MRSA develops that has homogeneously high methicillin

resistance.

Naimi et al. [13] conducted a prospective study of 1100

MRSA infections and found 131 (12%) CA-MRSA and 937

(85%) HA-MRSA strains (32 could not be classified). The CA-

MRSA isolates were more likely to be more susceptible to cip-

rofloxacin, clindamycin, erythromycin, and gentamicin than

were the HA-MRSA isolates ( ). However, most CA-P � .001

MRSA infections were initially treated with antimicrobials to

which the isolate was not susceptible. In a recent analysis by

LaPlante et al. [14], CA-MRSA isolates were found to be sig-

nificantly more susceptible to trimethoprim-sulfamethoxazole,

clindamycin, and ciprofloxacin than were HA-MRSA isolates

( ).P � .05

It is noteworthy, however, that some SCCmec types carry

various additional genetic elements (Tn554, which encodes re-

sistance to macrolides, clindamycin, and streptogramin B; and

pT181, which encodes resistance to tetracyclines) that can con-

fer resistance to additional antibiotic classes; these genetic el-

ements are especially common in HA-MRSA [12]. In addition,

strains that are resistant to erythromycin can quickly become

resistant to clindamycin because of the inducibility of resis-

tance. Inducible resistance to clindamycin is not detected by

routine susceptibility testing but requires the use of other meth-

ods, such as the D-test [15]. The standard disk diffusion (DD)

test is performed by placing a 15-mg erythromycin disk and a

2-mg clindamycin disk at a distance of 15–20 mm (closer than

the standard dispenser spacing of 26–28 mm). Flattening of the

clindamycin zone of inhibition in the area between the disks

to resemble the letter “D” indicates inducible resistance [15].

If these tests are not readily available, it is better not to use

clindamycin in these cases.

ADVANCES IN MRSA DETECTION METHODSAND SUSCEPTIBILITY TESTING

Recently, the British Society for Antimicrobial Chemotherapy,

the Hospital Infection Society, and the Infection Control

Nurses Association published guidelines for the laboratory

diagnosis and susceptibility testing of MRSA [16]. Routine

identification of S. aureus should be performed via tube co-

agulase tests or latex agglutination tests; routine use of mo-

lecular tests for identification is not currently practical for

most diagnostic laboratories. However, molecular testing may

be useful when there is a high index of suspicion for MRSA.

Susceptibility testing may be performed through any standard

recognized method, and latex methods to detect PBP 2a and/

or PCR tests to detect the mecA gene can provide confirmation

of equivocal results [16].

DD testing with cefoxitin has been well correlated with the

presence of mecA-mediated oxacillin resistance in S. aureus

and has excellent sensitivity (98%) and specificity (100%)

[17]. Swenson et al. [17] identified cefoxitin DD breakpoints

of �19 mm (resistant) and �20 mm (susceptible) for S. au-

reus. These authors also reported that the cefoxitin DD test

gave results equivalent to oxacillin broth microdilution tests

and oxacillin DD tests but was easier to interpret and did not

require transmitted light for identification of resistance. On

the basis of this study, the Clinical and Laboratory Standards

Institute (CLSI; formerly the National Committee for Clinical

Laboratory Standards) now recommends the use of the ce-

foxitin DD test for detecting methicillin resistance in S. au-

reus, and the method has been validated in an international

collection of staphylococci from the SENTRY antimicrobial

surveillance program [18].

Two novel molecular detection methods are worthy of men-

tion, although they are not ready for routine clinical use. The

3-dimensional microarray system described by Nagaoka et al.

[19] has a sensitivity 10-fold greater than that of standard PCR

methods and provides a high level of data reproducibility. It is

a rapid, specific, and easy test for the simultaneous detection

of resistance to levofloxacin and the presence of the mecA gene

in S. aureus. Zhang et al. [20] reported the development of a

novel multiplex PCR assay that both characterizes and subtypes

SCCmec in MRSA (e.g., types I–V and subtypes IVa–IVd) using

sets of SCCmec type- and subtype-unique and specific primers.

This method has demonstrated excellent (100%) sensitivity and

specificity in characterizing 54 known strains of MRSA with

various SCCmec types and subtypes. This tool may be useful

in understanding the epidemiology and clonal relatedness of

various MRSA isolates.

As is shown in figure 1, beneficial outcomes are associated

with rapid detection of MRSA. An interventional cohort study

of a new molecular MRSA screening test used at time of ad-

mission to the intensive care unit showed that 55 of 71 pre-

viously unknown MRSA carriers would have gone undetected

in the intensive care unit without screening. In addition, the

median time from intensive care unit admission to reporting

of test results was reduced from 4 days to 1 day ( ),P ! .001



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Figure 1. Time between intensive care unit admission and notificationof test results with standard culture versus rapid multiplex PCR. P !

for all comparisons between standard culture and rapid quick meth-.001icillin-resistant Staphylococcus aureus (qMRSA) screening. Values areexpressed as median. Data are from [21].

resulting in preemptive isolation and cohorting, thus reducing

MRSA cross-infections [21].

VANCOMYCIN NONSUSCEPTIBILITY IN MRSA

As is stated above, multidrug resistance is common in HA-

MRSA. It is worrisome that, in recent years, VISA have been

isolated with an increased incidence. Since 2002, 7 VRSA strains

have been isolated in the United States: these strains have not,

as far as we know, been reported elsewhere ([1] and M. Rybak,

personal communication, 2006). Multidrug-resistant strains of

both VISA and VRSA have been detected, suggesting that the

efficacy of antimicrobial agents for systemic infections such as

bacteremia, endocarditis, and osteomyelitis may soon be sig-

nificantly compromised [1]. The true incidence of hVISA

(strains ostensibly vancomycin susceptible as assessed by use

of current CLSI breakpoints but with a subpopulation able to

grow in the presence of vancomycin) is currently unknown

because of detection problems, which are detailed later in this

article.

An hVISA strain (Mu3) was first identified in 1996 in Japan

in a 64-year-old patient with MRSA pneumonia whose con-

dition responded poorly to vancomycin treatment [22]. This

strain had a vancomycin MIC of 4 mg/mL, which was initially

identified as an hVISA but would now be classified as a true

VISA according to current CLSI criteria. The first reported

strain of VISA (Mu50) was described in a 4-month-old Japanese

infant who was unsuccessfully treated with vancomycin: Mu50

was found to be an MRSA strain, with a vancomycin MIC of

8 mg/mL as assessed by broth microdilution [22]. In subsequent

years, close to 100 cases of S. aureus with reduced susceptibility

to vancomycin have been reported [1, 23, 24], with some strains

responsible for life-threatening systemic infections [23, 24].

Risk factors for VISA infection include recurrent MRSA infec-

tions treated with vancomycin and chronic renal failure. There

have been fewer cases of VRSA infection (7 cases reported), all

of which occurred in elderly US patients with multiple health

problems ([1] and M. Rybak, personal communication, 2006).

Susceptibility breakpoints established by CLSI for vancomycin

are currently as follows: susceptible, �2 mg/mL; intermediate

(VISA), 4–8 mg/mL; and resistant (VRSA), �16 mg/mL [25].

When effective, vancomycin inhibits cell wall synthesis. Resistant

staphylococci are able to maintain the cellular wall, and glyco-

peptides are unable to access cell wall synthesis sites.

The reduced vancomycin susceptibility of MRSA has been

attributed to, among other factors, functional loss of the ac-

cessory gene regulator (agr) operon under vancomycin selec-

tion pressure, especially with the agr group II phenotype. In

vitro studies have shown a greater propensity of agr group II

knockout strains to develop vancomycin heteroresistance, com-

pared with other agr groups [26]. In a study by Sakoulas et al.

[27], VISA and hVISA clinical isolates from diverse geographic

origins were found to be enriched in the agr group II poly-

morphism, in which several point mutations have been noted.

In another study, of 122 MRSA isolates from 87 patients treated

with vancomycin, Moise-Broder et al. [28] reported that the

conditions of 86% (31/36) of the patients infected with MRSA

that had the agr group II polymorphism did not to respond

to vancomycin. This polymorphism was found to be an in-

dependent predictor of vancomycin failure in patients with

MRSA infection ( ). This genetic marker may provideP p .005

a survival advantage to the MRSA strains, toward development

of reduced vancomycin susceptibility. However, this may not

be the only factor that plays a role.

Currently, hVISA and VISA strains are, in all likelihood,

being underreported because of nonstandard detection tech-

niques [23]. Typically, DD is the only method used in the

clinical laboratory; this may be acceptable for detection of

VRSA but is not satisfactory for detection of hVISA and VISA

strains [1]. Available automated methods, such as Vitek (bio-

Merieux) and Microscan (Dade Behring), do not identify all

known strains of VRSA adequately and should not be used for

the routine detection of hVISA and VISA [1]. A vancomycin

agar screening plate should be used. Existing commercially

available screening plates still contain a vancomycin concen-

tration of 6 mg/mL, on the basis of the old CLSI breakpoint of

�4 mg/mL. In view of the new lowered vancomycin suscepti-

bility breakpoint mentioned above, screening plates with a

lower vancomycin concentration would appear to be in order

and urgently require evaluation. In addition, routine screening

for glycopeptide resistance must be done when MRSA strains

are isolated, to detect such strains early and to prevent out-

breaks [24]. In all cases, at the very least, a vancomycin agar

screening test with a more appropriate vancomycin concentra-

tion (to be determined, but !6 mg/mL) should be used. A better



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Figure 2. Identification of glycopeptide resistance by macro Etest�. GISA, glycopeptide-intermediate Staphylococcus aureus; GSSA, glycopeptide-susceptible S. aureus; hGISA, heterogeneous glycopeptide-intermediate S. aureus; TP, teicoplanin; VA, vancomycin. Adapted from Bolmstrom et al.[30], with permission from AB BIODISK.

approach, in countries with the financial capability, is routine

testing of all MRSA strains with an overnight vancomycin Etest

strip (AB BIODISK), followed, if necessary, by a macro Etest

for all isolates with vancomycin MICs of 1–2 mg/mL isolated

from patients with S. aureus infections not responding to van-

comycin therapy [29]. As is depicted in figure 2, a macro Etest

comparing vancomycin and teicoplanin can easily differentiate

between VISA and hVISA: vancomycin susceptible when both

MICs are low, hVISA when the vancomycin MIC is low but

the teicoplanin MIC is high, and VISA when both MICs are

high [30]. It has been reported that teicoplanin susceptibility

in MRSA is lost before vancomycin susceptibility. The reason

for this is not known, but the lower baseline activity of tei-

coplanin for MRSA, compared with that of vancomycin, may

result in easier emergence of resistance to teicoplanin [31]. To

improve the detection of vancomycin or glycopeptide resistance

in MRSA, it is very important to consider the testing method.

The Etest may offer the best alternative, particularly because

not all of these strains will be detected by CLSI microbroth

dilution methodology [23].

CONCLUSIONS

The epidemiological and microbiological characteristics of

pathogenic organisms have been rapidly shifting because of

selection pressure. MRSA strains are now common both in the

health care setting and more widely in the community. Mul-

tidrug-resistant strains of MRSA are rapidly evolving, including

the more serious glycopeptide-nonsusceptible strains. Com-

mendable advances in delineating the genetic characteristics of

MRSA have recently been made, although this aspect is far

from being completely understood, and new questions are be-

ing raised—for example, the importance of PVL as a virulence

factor in CA-MRSA. Options for treating multidrug-resistant

S. aureus infection are few, and, as new strains emerge, the

options are becoming even more limited. Further study of

MRSA and evaluation of optimal practices to detect MRSA

infections clearly are needed. Active screening and detection of

glycopeptide nonsusceptibility in MRSA will almost certainly

result in more accurate representation of prevalences of hVISA

and VISA strains and, possibly, VRSA strains. A rise in systemic

infections caused by non–glycopeptide-susceptible S. aureus

strains will be a very serious development indeed, leaving the

clinician with very few therapeutic options.

Acknowledgments

Supplement sponsorship. This article was published as part of a sup-plement entitled “Bugging the Bugs: Novel Approaches in the StrategicManagement of Resistant Staphylococcus aureus Infections,” jointly spon-sored by the Dannemiller Memorial Educational Foundation and EmeritusEducational Sciences and supported by an educational grant from Ortho-McNeil, Inc., administered by Ortho-McNeil Janssen Scientific Affairs, LLC.



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Potential conflicts of interest. P.C.A. has received research/grant sup-port from Wyeth, Pfizer, Johnson & Johnson, AstraZeneca, Bayer, Repli-dyne, Affinium, Basilea, Arpida, Daiichi-Sankyo, and Novexel; is on thespeakers’ bureau for Wyeth; and is a consultant for Wyeth, Johnson &Johnson, and AstraZeneca.

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