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Diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus in Latin America.

INTRODUCTION

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of infections globally and a growing problem across Latin America. (1-3) Epidemiologic studies in the region have charted a significant rise in MRSA infections both in hospital and community settings. (1) A key step in the successful treatment of these infections is early and accurate diagnosis.

In clinical settings, diagnosis is based on a combination of epidemiologic information, clinical symptoms and characterization of the infecting MRSA strain. The Monitoring/Surveillance Network for Resistance to Antibiotics, set up with the support of the Pan-American Health Organization (PAHO), provides epidemiologic information on bacterial resistance across Latin America. In some countries, including Argentina, Chile, Ecuador, Uruguay and Venezuela, an organized quality control system is present to support local surveillance, but in others, the capacity for microbiologic diagnosis is limited to a few large university hospitals in the major cities, and limited data, especially regarding community-acquired MRSA, are available in these regions.

Various international guidelines are available that provide recommendations for best practices in MRSA diagnosis and treatment. However, adoption of these recommendations can be sporadic, especially at regional levels where resources may be a significant limiting factor. Most guidelines provide a range of options for MRSA diagnosis that can be adapted for different regional requirements. However, it may not always be clear which tests are appropriate and sufficient in specific circumstances. Additional guidance is therefore required to establish consistency of approach across the region.

GUIDELINES FOR DIAGNOSIS OF MRSA

Guideline documents have been published in a number of countries outlining recommended protocols and procedures for the identification of MRSA (Table 1 (4-9)).

The Clinical and Laboratory Standards Institute (CLSI; formerly the National Committee on Clinical Laboratory Standards, NCCLS) in the USA has developed a range of best practice documents covering all aspects of microbiologic testing, including recent publications entitled 'Performance Standards for Antimicrobial Susceptibility Testing' (4) and 'Surveillance for Methicillin-Resistant Staphylococcus aureus: Principles, Practices, and Challenges'. (5)

The European Antimicrobial Resistance Surveillance System (EARSS), funded by the European Commission, provides a comprehensive surveillance and information system on the spread of antimicrobial resistance in Europe. EARSS has published protocols for diagnostic testing of various organisms with antibiotic resistance traits, including MRSA, VISA and VRSA. (6,10) Similarly, the Sociedad Espanola de Infectologia y Microbiologia Clinica (SEIMC), based in Spain, has published recommendations for the identification of various bacterial strains with antimicrobial resistance, including MRSA. (7)

In the UK, the British Society for Antimicrobial Chemotherapy (BSAC) published their first guidelines on microbial sensitivity testing in 1991, including minimum inhibitory concentration (MIC) breakpoints for clinically relevant bacteria, and more recently provided standardized methods for disc susceptibility testing for a range of organisms, including MRSA. (8) A Joint Working Party of the BSAC, the Hospital Infection Society (HIS) and the Infection Control Nurses Association (ICNA) recently published evidence-based guidelines on the laboratory diagnosis of MRSA. (9) These guidelines include recommendations on the identification of MRSA and methods of susceptibility testing and screening.

Since the various guidelines differ in their scope and detail, and generally do not apply specifically to infections in Latin America, infection control teams are advised to choose guidelines to follow and to adapt them to their local situation, considering such factors as epidemiology, available antibiotics and resources, likely sources of infection, and risk factors associated with their specific patient population and environment. The CLSI guidelines are the guidelines of choice in most Latin American countries. Evaluation of the implementation of guidelines is also important, as is education of healthcare workers, in order to ensure that consistent best practices are maintained.

IDENTIFICATION OF S. AUREUS

S. aureus causes a wide range of clinical infections, resulting in direct invasion of bacteria into different organs and consequent tissue damage. The clinical manifestations of infection result from the release of various toxins, either locally or systemically, and include a range of diseases dependent on the location of the infection (Table 2).

For localized infections, a clinical diagnosis is often sufficient without the need for analysis of cultures. However, for systemic infections, proper and prompt detection of S. aureus strains and their susceptibility to different antibiotics is of paramount importance in order for healthcare workers to provide appropriate treatment, and to initiate relevant control measures.

Initial, rapid assessment of clinical samples is typically achieved using conventional microscopy, by which staphylococci appear as rounded, Gram-positive cocci growing in clusters. It is important to distinguish S. aureus isolates from other staphylococcal species, such as coagulase-negative staphylococci (CoNS), and various tests are available to achieve this (Table 3). While several of these tests can be used interchangeably under appropriate circumstances, the relative benefits and limitations of each should be understood by microbiologists and healthcare professionals in order that appropriate conclusions can be drawn.

A number of factors influence the choice of S. aureus identification tests employed in a given situation, including cost, speed of result, facilities available, sensitivity and specificity. The joint BSAC/HIS/ICNA guidelines (9) recommended that a tube coagulase or latex agglutination test should be used for routine identification of S. aureus or for confirmation after DNase tests, or after negative results in a slide coagulase test. Although the readout from the slide test is much quicker than for the tube test (15 s vs. 4-24 h), the former has a higher false-negative rate (~15%). Consequently, the tube test is considered more definitive, and is the preferred coagulase test for identification of S. aureus.

Under circumstances where clinicians require a rapid assessment of MRSA, a slide coagulase test may be confirmed by latex agglutination, automated approaches or molecular tests. An international multicenter study, in which various commercial agglutination kits for identification of S. aureus were assessed using 892 staphylococcal isolates, found reliable detection of S. aureus (> 98% sensitivity and > 98% selectivity for Slidex Staph Plus). (11) Automated tests provide a similar level of accuracy for identification of S. aureus and are used across Latin America, but these may not be available in smaller local centers.

More sophisticated approaches to speciation of staphylococci are available that can provide identification of most species, but tend to involve a greater battery of tests. In a Brazilian study, Iorio et al. (12) demonstrated a scheme for the rapid identification of 198 staphylococcal isolates (including 17 of S. aureus) using a simplified battery of phenotypic tests. Staphylococci were initially identified using Gram stain, the catalase test, acid production from glucose in Hugh and Leifson's OF base medium, and susceptibility to bacitracin. Nine phenotypic tests were then used to distinguish staphylococcal species, achieving 98.5% accuracy across species (100% for S. aureus) in 72 h. Such schemes may be useful in routine laboratories, and particularly in developing countries where costs and resources are significant issues.

Methicillin sensitivity testing

There are many options for testing methicillin susceptibility of S. aureus, including disc diffusion, MIC measurements (in broth or by Etest), chromogenic agar, latex agglutination, automated methods, rapid screening methods and molecular approaches (see Table 4 (4,5,8,9,13-20) for details).

For media-based methods, test conditions such as media type, incubation times and temperature, play an important role in determining the outcome of methicillin sensitivity tests, as reflected in many of the published guidelines, and these factors should be considered carefully when designing appropriate tests. The BSAC recommends Columbia or Mueller Hinton agar supplemented with NaCl (2%) for dilution and disc diffusion methods, (4,8) and addition of up to 5% NaCl to media has been shown to improve detection of resistance for most strains. (21,22) Typically, methicillin resistance is detected more reliably at lower temperatures (30-35[degrees]C), (23-25) although some rare strains may grow slowly at 30[degrees]C when 5% NaCl is present. Both the CLSI and BSAC recommend that incubations are performed for 24 h, (4,8) but for some heterogeneous strains, resistant sub-populations may grow more slowly, and incubations of 48 h may be required to improve detection. Cefoxitin has now taken over as the antibiotic of choice for methicillin sensitivity testing, with methicillin itself no longer produced. Oxacillin remains a second option, but several publications have demonstrated that cefoxitin is more reliable than oxacillin. (13,14,26,27)

In Latin America, the methodology used for identification of MRSA differs between countries. The disc diffusion method, using oxacillin or cefoxitin discs, is popular in some countries, whereas Etest strips are generally considered too expensive for routine use. Confirmation tests, such as the methicillin screen plate test, are not widely used and molecular analysis of MRSA strains is restricted to a few centers in Brazil, Argentina, Chile, Mexico and Colombia. In cases of nosocomial outbreaks, the identity of MRSA strains is usually assumed from the phenotypic pattern of antibiotic resistance. Many laboratories in Latin America use automated methods, and these offer a convenient and, in some cases, rapid approach to identification of MRSA. The VITEK GPI system and more recent VITEK2 (Biomerieux[R]), the Microscan[R] Rapid POS COMBO (Dade/Microscan) and the Phoenix system (BD Biosciences), are all widely used for detection of MRSA, and the Vitek system will also soon include a screening test for vancomycin susceptibility.

The joint BSAC/HIS/ICNA guidelines (9) recommended that "a standard, recognized method, such as those published by the BSAC or the CLSI, should be used for routine susceptibility testing of S. aureus', but that 'other tests should be considered acceptable if they give equivalent or better performance". Disc diffusion, MIC determination and latex agglutination are all sufficient and affordable methods for routine methicillin sensitivity testing. Latex agglutination to detect penicillin-binding protein 2a (PBP2a) may also be used as a confirmatory method.

Rapid detection of MRSA is especially important in settings where quick preventive or therapeutic measures are needed, such as in intensive care units and in some surgical interventions where prosthetic material substitutions are required. Latex agglutination and ChromAgar are reliable methods for detection of MRSA and the results are available more quickly than other methods.

Importantly, consistent protocols should be introduced for all of the above tests where possible, and should be carried out using appropriate susceptible and resistant control strains, such as those outlined in the BSAC and CLSI guidelines. (4,8) For MIC and disc diffusion studies, reference values for MIC and zone of inhibition are provided in the CLSI guidelines to define susceptibility, intermediate resistance and resistance to specific antimicrobial agents (Table 5). (4) MRSA should be reported as resistant to all currently-available -lactam agents (penicillins, -lactamase/-lactamase inhibitor combinations, cephems and carbapenems), since activity of -lactam agents against MRSA in in vitro tests does not necessarily translate into clinical efficacy.

DETECTION OF REDUCED SUSCEPTIBILITY TO VANCOMYCIN (VRSA AND VISA)

MRSA infections are commonly treated with glycopeptide antibiotics such as vancomycin and teicoplanin. However, MRSA isolates with reduced susceptibility or resistance to vancomycin have emerged in recent years, (28) including in Latin America. (29) Globally, these isolates have been termed vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) depending on their level of resistance. Although VISA/VRSA strains have not been identified frequently in Latin America, and the incidence does not appear to be increasing, (30) the potential importance of these organisms is reflected in the inclusion of vancomycin susceptibility testing within guidelines for the diagnosis of MRSA. (8-10)

The current 'gold standard' for the diagnosis of VISA or VRSA is the screen test. Here, plates made up of brain-heart infusion agar and 6 mg/mL vancomycin are spotted with a 10 [micro]l inoculum of a 0.5 McFarland bacterial suspension and incubated for 24 h, with the growth of more than one colony signifying a positive result. (31) S. aureus ATCC 25923 and Enterococcus faecalis ATCC 51299 may be used as negative and positive controls, respectively. Mueller Hinton agar containing vancomycin or teicoplanin may also be used in the screen test, (8,9) but a longer incubation time (48 h) is suggested.

Most guidelines recommend MIC methods for confirmation of positive screen test results. (9,10) However, care should be taken in choosing appropriate tests for VISA and VRSA, since not all methods are appropriate for both strains (Table 6). VISA and VRSA, for example, are not reliably detected using automated methods, (31,32) whereas disc diffusion is inappropriate for VISA, but can be used for VRSA. Generally, a non-automated MIC method (e.g. broth dilution, agar dilution or Etest) with a 24-hour incubation is appropriate. (8,10) Strains with a MIC of [less than or equal to] 2 [micro]g/ mL are considered susceptible to vancomycin (Table 5), (4) although increasing vancomycin MICs within this 'susceptible' range have been linked to an increased risk of clinical failure. (33) VISA with heterogeneous sensitivity to vancomycin (h-VISA) should be confirmed by a population analysis profile (PAP) method, since MICs for these strains may be similar to those for susceptible strains. (9)

It has been proven that h-VISA significantly complicates the treatment of bacteremia patients and that it is frequently not identified by clinical laboratories. The best detection method for h-VISA is the measurement of the area under the curve (AUC) from a PAP test, however it is very labour-intensive, costly and is not appropriate in a clinical setting. There are currently three reasonable alternatives to PAP that are highly sensitive and specific and that must be used in all MRSA isolates with a vancomycin MIC of 1-2 [micro]g/mL.

1) "New strip" Etest detection method for resistance to glycopeptides (vancomycin, 32-0.5 [micro]g/mL; teicoplanin, 32-0.5 [micro]g/mL; bioMerieux AB) (34)

2) Mueller-Hinton agar supplemented with 5 [micro]g/mL teicoplanin

3) Plates made up of brain heart infusion agar supplemented with 6 [micro]g/mL vancomycin, as described in the literature (28,35)

Following positive tests for VISA or VRSA, samples should be forwarded to a reference laboratory for population analysis using appropriate control strains (for example, ATCC 700698, ATCC700699 and Oxford strain, or an alternative control (10)). Organisms that can be used as controls for sensitivity testing, for the evaluation of low levels of glycopeptide resistance (glycopeptide intermediate S. aureus [GISA]) and heterogeneous glycopeptide resistance (heterogeneous GISA [h-GISA]) are: S. aureus ATCC 29213, ATCC 700698 (Mu3; h-GISA) and ATCC 700699 (Mu50; GISA).

The heteroresistance phenomenon has been in MRSA strains which, despite having a vancomycin MIC below that of the breakpoint of susceptible strains, had subpopulations growing in the presence of 4-8 [micro]g/mL of vancomycin. (28,36) Since their description, these strains have been called vancomycin-heteroresistant and have been detected in several countries, (37,38) including some countries in Latin America. In a study carried out in Venezuela, Colombia, Ecuador and Peru, Reyes et al. described nine strains of h-VISA in 1,570 S. aureus (0.57%). (39) This heterogeneity in vancomycin resistance is similar to that described for methicillin in MRSA, where only 1x[10.sup.-6] bacteria express this characteristic.

Vancomycin heteroresistance has been considered as a potential cause of therapeutic failure. (38) However, to know the real incidence and importance of this strain type, it is necessary to establish a reliable and reproducible detection method. Some authors suggest that current vancomycin heteroresistance detection methods induce, rather than detect, resistance to vancomycin, and thus it will be impossible to establish its clinical relevance until we better understand the control mechanisms of vancomycin resistance. (40,41)

Susceptibility testing should also be performed for erythromycin, clindamycin (including detection of the inducible mechanism in erythromycin resistant strains), daptomycin, linezolid, rifampin, quinupristin/dalfopristin and trimethoprim/sulfamethoxasole. These tests can be performed in-house if appropriate facilities are available or, more frequently, in a reference laboratory. It is recommended that probable isolates of VISA and VRSA are sent to a reference laboratory as quickly as possible, even if there is a capability to test additional agents in-house, in order to facilitate organism confirmation and enhance infection control efforts.

IMPLICATIONS FOR THE REGION

The growing incidence and awareness of MRSA across Latin America has been met by an extensive effort towards early diagnosis, appropriate intervention and widespread surveillance. As would be expected from a region with such diversity in resources, a wide variety of diagnostic tests are used routinely in clinical practice, illustrated in this review, and various quality control measures are applied. As MRSA is likely to be a continuous threat to public health in Latin America for the foreseeable future, it is timely that current processes are reviewed and measures to ensure consistent practices are adopted across the region.

Existing guidelines covering MRSA diagnosis and treatment are thorough, and these should be used as a basis to standardize practices. A tiered set of recommendations may be required to accommodate both well-funded, larger centers, and local clinics with limited resources. Also, recommendations may need to be adapted for individual countries based on local resources, epidemiology and specific clinical requirements.

A coordinated system for quality control is a key requirement for successful MRSA diagnosis, and centralized, accessible reference facilities should be developed to support local centers. Collaboration within individual countries and across the region is important in this regard.

Education is a key factor in providing consistency of approach. Microbiology laboratories should participate in the education of medical and healthcare students and workers to perform procedures appropriately, and regional support networks should be set up to provide longer-term support and to facilitate the introduction of new techniques. Finally, systems to evaluate the implementation of guidelines should be introduced in order to ensure that consistent and best practices are adopted and maintained across the region.

While these recommendations are unlikely to halt the spread of antibiotic resistant S. aureus strains across Latin America, they should assist healthcare workers in achieving the most appropriate balance between the management of local resources and the provision of high quality diagnostics, both in hospitals and in the local communities.

IMPLICATIONS FOR CLINICAL PRACTICE

* Diagnosis of MRSA should be carried out according to specific guidelines.

* Guidelines should be selected and adapted to take account of local resources and needs.

* A coordinated system for quality control should be established to support microbiologic diagnosis, including regional access to central reference facilities.

* Medical and healthcare professionals should be educated in best practice diagnosis.

* Systems should be established to evaluate the implementation of guidelines and best practices across Latin America.

ACKNOWLEDGEMENTS

Financial support

Pfizer Inc., New York, NY, USA, provided support for meetings of the Latin American Working Group on Gram Positive Resistance. Pfizer Inc. had no involvement in the collection, analysis and interpretation of data, in the writing of the manuscripts, or in the decision to submit the articles for publication.

Manuscript preparation

The support provided by Choice Pharma (Hitchin, UK), funded by Pfizer Inc., consisted of manuscript formatting and writing assistance.

DISCLOSURES

J. Zurita: Advisory Board member and consultant for Pfizer; received research grant from Wyeth.

C. Mejia: Advisory Board member for Pfizer and Abbott; consultant for Pfizer; received funding from Tibotec for HIV research, from Avexa for studies in HIV treatment and from Merck for participation in the SMART study.

M. Guzman-Blanco: Advisory Board member for Pfizer, Merck and BD; consultant for Pfizer, Wyeth and Janssen; received research funding from Wyeth and Merck.

REFERENCES

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(13.) Cauwelier B, Gordts B, Descheemaecker P, Van Landuyt H. Evaluation of a disk diffusion method with cefoxitin (30 microg) for detection of methicillin-resistant Staphylococcus aureus. Eur J Clin Microbiol Infect Dis. 2004; 23(5):389-92.

(14.) Skov R, Smyth R, Clausen M et al. Evaluation of a cefoxitin 30 microg disc on Iso-Sensitest agar for detection of methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother. 2003; 52(2):204-7.

(15.) Huang MB, Gay TE, Baker CN et al. Two percent sodium chloride is required for susceptibility testing of staphylococci with oxacillin when using agar-based dilution methods. J Clin Microbiol. 1993; 31(10):2683-8.

(16.) Novak SM, Hindler J, Bruckner DA. Reliability of two novel methods, Alamar and E test, for detection of methicillin-resistant Staphylococcus aureus. J Clin Microbiol. 1993; 31(11):3056-7.

(17.) Petersson AC, Miorner H, Kamme C. Identification of mecA-related oxacillin resistance in staphylococci by the E test and the broth microdilution method. J Antimicrob Chemother. 1996; 37(3):445-56.

(18.) Weller TM, Crook DW, Crow MR et al. Methicillin susceptibility testing of staphylococci by Etest and comparison with agar dilution and mecA detection. J Antimicrob Chemother. 1997; 39(2):251-3.

(19.) Perry JD, Rennison C, Butterworth LA et al. Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus. J Clin Microbiol. 2003; 41(12):5695-8.

(20.) Hedin G, Fang H. Evaluation of two new chromogenic media, CHROMagar MRSA and S. aureus ID, for identifying Staphylococcus aureus and screening methicillin-resistant S. aureus. J Clin Microbiol. 2005; 43(8):4242-4.

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(30.) Picao R, Sader H, Jones R et al. Analysis of resistance and vancomycin 'reverse creep' in Latin America Staphylococcus aureus: ten-year report of the SENTRY Antimicrobial Surveillance Program (1997-2006). Clin Microbiol Infect. 2008; 14:S173.

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(34.) Yusof A, Engelhardt A, Karlsson A et al. Evaluation of a new Etest vancomycin-teicoplanin strip for detection of glycopeptide-intermediate Staphylococcus aureus (GISA), in particular, heterogeneous GISA. J Clin Microbiol. 2008; 46(9):3042-7.

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(36.) Hiramatsu K, Hanaki H, Ino T et al. Methicillin-resistant Staphylococcus aureus clinical strain with reduced vancomycin susceptibility. J Antimicrob Chemother. 1997; 40(1):135-6.

(37.) Marchese A, Balistreri G, Tonoli E et al. Heterogeneous vancomycin resistance in methicillin-resistant Staphylococcus aureus strains isolated in a large Italian hospital. J Clin Microbiol. 2000; 38(2):866-9.

(38.) Ariza J, Pujol M, Cabo J et al. Vancomycin in surgical infections due to methicillin-resistant Staphylococcus aureus with heterogeneous resistance to vancomycin. Lancet. 1999; 353(9164):1587-8.

(39.) Reyes J, Rincon S, Diaz L et al. Dissemination of methicillin-resistant Staphylococcus aureus USA300 sequence type 8 lineage in Latin America. Clin Infect Dis. 2009; 49(12):1861-7.

(40.) Howe RA, Wootton M, Walsh TR et al. Expression and detection of hetero-vancomycin resistance in Staphylococcus aureus. J Antimicrob Chemother. 1999; 44(5):675-8.

(41.) Boyle-Vavra S, Berke SK, Lee JC, Daum RS. Reversion of the glycopeptide resistance phenotype in Staphylococcus aureus clinical isolates. Antimicrob Agents Chemother. 2000; 44(2):272-7.

Jeannete Zurita [1] Carlos Mejia [2] Manuel Guzman-Blanco [3] on behalf of the Latin American Working Group on Gram Positive Resistance.

[1] Hospital Vozandes, Quito, Ecuador.

[2] Hospital Roosevelt, Guatemala City, Guatemala.

[3] Centro Medico de Caracas, Caracas, Venezuela.

Correspondence to: Jeannete Zurita Directora del Servicio de Microbiologia y Tuberculosis Hospital Vozandes Villalengua Oe2-37 Quito, Ecuador

Phone: +593-2-2262142

Fax: +593-2-2269234

E-mail: jzurita@hcjb. org.ec
Table 1. Guidelines for diagnosis of MRSA (4-9)

Source                      Guidelines

Clinical and Laboratory     Performance Standards
Standards Institute         for Antimicrobial
(CLSI)                      Susceptibility
                            Testing (4)

                            Surveillance for
                            Methicillin-Resistant
                            Staphylococcus
                            aureus: Principles,
                            Practices, and
                            Challenges (5)

European Antimicrobial      New and updated protocols
Resistance Surveillance     for antimicrobial
System (EARSS)              susceptibility
                            testing of pathogens
                            under EARSS
                            surveillance 2005 (6)

Sociedad Espanola           Protocolos de diagnostico
de Infectologia y           en Microbiologia (7)
Microbiologia
Clinica (SEIMC)

British Society for         BSAC standardized disc
Antimicrobial               susceptibility
Chemotherapy (BSAC)         testing method
                            (version 7) (8)

Joint Working Party of      Guidelines for the
the British Society         laboratory diagnosis
for Antimicrobial           and susceptibility
Chemotherapy, the           testing of
Hospital Infection          methicillin-resistant
Society and the            Staphylococcus aureus (MRSA) (9)
Infection Control
Nurses Association
(BSAC/HIS/ICNA)

Table 2. Clinical manifestations of
S. aureus infection

Source of infection         Disease

Skin and soft tissue        Impetigo, boils, carbuncles,
                            abscesses, cellulitis,
                            fasciitis, pyomyositis,
                            surgical and traumatic
                            wound infections

Foreign body-associated     Intravascular catheter,
                            urinary catheter

Intravascular               Bacteraemia, sepsis, septic
                            thrombophlebitis,
                            infective carditis

Bone and joints             Septic osteomyelitis,
                            septic arthritis

Respiratory                 Pneumonia, empyema,
                            sinusitis, otitis media

Other invasive infections   Meningitis, surgical
                            space infection

Toxin-mediated disease      Staphylococcal toxic
                            shock, food poisoning,
                            staphylococcal scalded
                            skin syndrome, bullous
                            impetigo, necrotizing
                            pneumonia, necrotizing
                            osteomyelitis

Table 3. Methods for identification of S.
aureus isolates

Principle                          Method

Test: Coagulase

Coagulase produced by              Tube test
certain Gram-positive              Detects free coagulase
cocci, including S.
aureus either in bound             Bacterial suspension
form (attached to the              added to diluted
bacterial cell wall)               rabbit plasma and
or as free enzyme,                 incubated at 37[degrees]C
converts fibrinogen to
insoluble fibrin in                Free coagulase causes
the presence of plasma,            plasma to clot into
resulting in clotting              a gel, usually within 4 h

Presence of coagulase              Slide test Detects
distinguishes S. aureus            cell-bound coagulase
from coagulase-negative
staphylococci (CoNS)               Bacterial suspension
                                   mixed with plasma on a slide

                                   Presence of bound
                                   coagulase causes
                                   cocci to clump
                                   together rapidly
                                   (5-10 s)

Test: Latex agglutination

Proteins on the surface            S. aureus bacterial
of S. aureus (e.g.                 suspension mixed with
protein A, clumping factor         specific latex spheres
group-specific antigens,
capsular polysaccharides),         Aggregation results
are recognized by                  in a clearing of
specific latex spheres,            background media
resulting in aggregation

Test: Catalase

S. aureus produce abundant         3% hydrogen peroxide
catalase, which can                added to colonies
interact with hydrogen             on agar plate
peroxide to produce oxygen
                                   Catalase-positive
                                   colonies produce
                                   oxygen and bubble
                                   almost immediately

Test: Mannitol salt agar (MSA)

S. aureus strains grow             Bacteria plated onto
well in high salt and              MSA consisting of
are able to ferment                mannitol, ~ 7.5-10%
mannitol to produce acid,          salt and the pH
which can be detected              indicator phenol
using a pH indicator               red

                                   Pathogenic staphylococci,
                                   such as S. aureus, grow
                                   well in high salt
                                   and turn MSA yellow
                                   through the release
                                   of acid

Test: Polymyxin B and novobiocin susceptibility

Polymyxin B is a cationic          Discs of polymyxin B
detergent antibiotic-specific      (10 U) added to agar
for Gram-negative bacilli          plate

                                   S. aureus are resistant to
                                   polymyxin B

Novobiocin is an amino-coumarin    Discs of novobiocin
antibiotic which can be            (5 [micro]g) added
used to differentiate S.           to agar plate
aureus from some CoNS
                                   S. aureus and some CoNS
                                   (e.g. S. epidermis)
                                   are sensitive to
                                   novobiocin, whereas
                                   most other CoNS (e.g.
                                   S. saprophyticus) are
                                   resistant

Test: DNase and heat-stable nucleases

Identification of S. aureus        Methods include
based on detection of              anti-nuclease test
specific DNase and                 to measure antibody
heat-stable nucleases              to nucleases common
common to S. aureus strains        to all strains of S.
                                   aureus, and metachromatic
                                   agar diffusion for
                                     heat-stable  nucleases

Test: Automated methods

Automated systems employ           Commercial systems
a battery of tests to              include: VITEK/VITEK2
achieve S. aureus                  (bioMerieux[TM])
identification
                                   Phoenix (BD Biosciences)

                                   Microscan (Dade Behring)
Test: Molecular methods

S. aureus contain a number         Methods include PCR
of genes that allow                (simple, multiplex
them to be distinguished           and real-time), DNA
from other staphylococcal          sequencing, and
species                            hybridization-based
                                   techniques

                                   Species-specific genes
                                   commonly used include
                                   nuclease (nuc),
                                   coagulase (coa), protein
                                   A (spa), femA and
                                   femB, Sa442, and S.
                                   aureus specific 16S
                                   rRNA

Principle                          Considerations

Test: Coagulase

Coagulase produced by              Tube test
certain Gram-positive
cocci, including S.                Some staphylococcus
aureus either in bound             species not commonly
form (attached to the              found in human isolates (e.g. S.
bacterial cell wall)               schleiferi and S.
or as free enzyme,                 intermedius) may
converts fibrinogen to             test positive
insoluble fibrin in
the presence of plasma,            Occasionally, rare
resulting in clotting              S. aureus may test negative

                                   Some strains require up
                                   to 24 h incubation

                                   Tests should not
                                   be incubated beyond
                                   24 h

Presence of coagulase              Slide test
distinguishes S. aureus
from coagulase-negative            Rapid result
staphylococci (CoNS)               10-15% of S. aureus
                                   strains test negative

                                   Some other staphylococcus
                                   species test positive
                                   (e.g. S. schleiferi,
                                   S. lugdunensis)

Test: Latex agglutination

Proteins on the surface            Latex agglutination
of S. aureus (e.g.                 kits specific for
protein A, clumping factor         S. aureus available
group-specific antigens,           commercially (e.g.
capsular polysaccharides),         Staphaurex, Staphaurex
are recognized by                  Plus; Wellcome Diagnostics,
specific latex spheres,            UK)
resulting in aggregation
                                   Detect both MSSA and MRSA

                                   Sensitivity > 98% for all S.
                                   aureus strains

                                   Cheap, rapid test

                                   Some other
                                   staphylococcus species
                                   may test positive (e.g.
                                   S. schleiferi, S.
                                   lugdunensis)

Test: Catalase

S. aureus produce abundant         Cheap, rapid result
catalase, which can
interact with hydrogen             Distinguishes catalase-producing
peroxide to produce oxygen         cocci (e.g. staphylococci)
                                   from non-producers
                                   (e.g. streptococci)

                                   Cannot be performed
                                   on blood agar because
                                   blood contains catalase

Test: Mannitol salt agar (MSA)

S. aureus strains grow             Usually detects MRSA
well in high salt and              and MSSA MSA can
are able to ferment                be supplemented
mannitol to produce acid,          with other antimicrobials,
which can be detected              including cefoxitin,
using a pH indicator               for MRSA screening

Test: Polymyxin B and novobiocin susceptibility

Polymyxin B is a cationic          Polymyxin B and
detergent antibiotic-specific      novobiocin discs
for Gram-negative bacilli          are relatively
                                   cheap and easy
                                   to use

                                   They should be used
                                   as part of a
                                   series of diagnostic
                                   tests for S. aureus

Novobiocin is an amino-coumarin
antibiotic which can be
used to differentiate S.
aureus from some CoNS

Test: DNase and heat-stable nucleases

Identification of S. aureus        Some rare CoNS can be
based on detection of              positive in heat-stable
specific DNase and                 nuclease tests
heat-stable nucleases
common to S. aureus strains        S. aureus can be detected
                                   on deoxyribonuclease (DNase)
                                   plates used to screen isolates

                                   Since various amounts of
                                   DNase are produced by
                                   CoNS, positives should
                                   be confirmed with an
                                   additional test

Test: Automated methods

Automated systems employ           Convenient and reliable
a battery of tests to              for S. aureus
achieve S. aureus
identification                     Resource issues limit use in
                                   smaller laboratories

Test: Molecular methods

S. aureus contain a number         Robust and simple to
of genes that allow                perform
them to be distinguished
from other staphylococcal          Many local laboratories do not
species                            have facilities for molecular
                                   approaches

Table 4. MRSA susceptibility testing (4,5,8,9,13-20)

Principle                       Recommended method

Test: Disc diffusion
(Kirby-Bauer)

Discs containing                Disc diffusion (4,8,9)
antimicrobial agent
placed on the surface           2% NaCl in MH or Columbia agar
of agar plate inoculated
with bacterial                  Incubation 30-35[degrees]C for
suspension, and zone            [greater than or equal to] 24 h
of inhibition assessed
around colonies                 Cefoxitin discs are more reliable
                                than oxacillin discs. (13,14,26,27)
                                Zone for oxacillin or
                                cefoxitin should be
                                read by holding
                                plate up to light
                                source

Test: MIC determinations
following antibiotic
dilution in broth or agar

Growth in different             Broth (4)
dilutions of antimicrobial
agent in broth or agar          2% NaCl in MH broth + oxacillin
to reveal MIC                   or cefoxitin

                                Inoculum of 5x[10.sup.5] cfu/mL

                                Incubation 33-35[degrees]C
                                for 24 h

                                Agar (4,8,9)

                                Tests on MH or Columbia
                                agar with 2%

                                NaCl + oxacillin

                                Inoculum of [10.sup.4]
                                cfu/mL

                                Incubation at 30-35
                                [degrees]C for 24 h

Test: Etest

Etest[R] (bioMerieux),          Etest (15-18)
based on plastic
strips containing a             2% NaCl in MH agar
predefined gradient
of 15 antibiotic                Inoculum density
concentrations                  equivalent to 0.5-1.0
                                McFarland standard

                                Incubation at 35[degrees]C
                                for 24 h

                                Strips laid out on
                                agar plate, and growth
                                assessed along
                                gradient to determine MIC

Test: Plate screening

Agar plates for                 Plate screening (4)
screening
                                4% NaCl in MH agar +
                                oxacillin 6 mg/L

                                Inoculum density
                                equivalent to 0.5

                                McFarland standard

                                Incubation at [less than
                                or equal to] 35[degrees]C
                                for 24 h

                                > 1 colony is indicative
                                of resistance

Test: Latex agglutination
detection of PBP2a

MRSA strains produce            PBP2a extracted from a
penicillin-binding              suspension of colonies
protein 2a (PBP2a)
                                Latex particles coated
                                with monoclonal
                                antibodies to PBP2a
                                added to extract

                                Presence of PBP2a
                                causes particle
                                agglutination

                                Addition of a penicillin
                                may induce PBP2a
                                production and give
                                a stronger reaction

                                Also available in
                                slide format

Test: Chromogenic agar

Chromogenic agars               Several commercial agars
specific for MRSA,              available, (19,20)
allowing rapid detection        including:
of colonies through
colored reactions               S. aureus ID (bioMerieux)

                                CHROMagar MRSA (CHROM agar)

                                Chromogenic MRSA Agar (Oxoid)

Test: Automated methods

Automated systems employ        Commercial systems
a battery of tests
to provide a convenient,        VITEK GPI (bioMerieux[R])
and often rapid,
approach to S. aureus           Phoenix (BD Biosciences)
and MRSA
identification                  Microscan (Dade Behring)

Test: Molecular methods

MRSA strains possess mecA       Methods include PCR
gene, which encodes             (simple, multiplex
penicillin-binding              and real-time), DNA
protein 2a (PBP2a)              sequencing and
                                hybridization-based
                                techniques

                                Detection of mecA for
                                methicillin resistance
                                is combined with
                                species-specific
                                genes such as nuclease
                                (nuc), coagulase
                                (coa), protein A
                                (spa), femA and femB,
                                Sa442, 16S rRNA
                                fibrinogen-binding
                                protein genes

Principle                       Considerations

Test: Disc diffusion
(Kirby-Bauer)

Discs containing                Some strains show
antimicrobial agent             heteroresistance, which
placed on the surface           is apparent as an inner
of agar plate inoculated        zone within a larger
with bacterial                  zone of inhibition
suspension, and zone
of inhibition assessed          On oxacillin discs, some
around colonies                 hyper-producers of
                                penicillinase give no
                                zone of inhibition
                                and will be incorrectly
                                reported as MRSA

Test: MIC determinations
following antibiotic
dilution in broth or agar

Growth in different             Relatively cheap
dilutions of antimicrobial
agent in broth or agar          More cumbersome than disc
to reveal MIC                   diffusion, Etest and latex
                                agglutination methods

Test: Etest

Etest[R] (bioMerieux),          Easy to set up
based on plastic
strips containing a             Not widely used in Latin America due
predefined gradient             to cost
of 15 antibiotic
concentrations

Test: Plate screening

Agar plates for                 Cheap, reliable
screening
                                Recommended for screening and
                                confirming resistance
                                identified by disc
                                diffusion

Test: Latex agglutination
detection of PBP2a

MRSA strains produce            Rapid, cheap and reliable
penicillin-binding              method for detection of MRSA
protein 2a (PBP2a)
                                Suitable for confirmation of
                                resistance

                                Isolates producing small
                                amounts of PBP2a may give
                                weak agglutination
                                reactions, or agglutinate
                                slowly

                                Some rare MRSA isolates
                                may test negative

                                May not be reliable
                                for colonies grown on
                                media containing NaCl

Test: Chromogenic agar

Chromogenic agars               Chromogenic agars can
specific for MRSA,              have high sensitivity
allowing rapid detection        for MRSA (> 98%)
of colonies through
colored reactions               Appropriate for rapid MRSA screening

Test: Automated methods

Automated systems employ        Convenient and reliable
a battery of tests              for S. aureus
to provide a convenient,
and often rapid,                Some false-positives may be identified
approach to S. aureus
and MRSA                        These methods can be used for
identification                  detection of MRSA, but do not
                                detect VRSA

Test: Molecular methods

MRSA strains possess mecA       Robust and simple to perform
gene, which encodes
penicillin-binding              Rapid and unambiguous
protein 2a (PBP2a)              characterization of MRSA

                                Reference method of
                                choice for MRSA confirmation

                                May detect occasional
                                susceptible strains
                                carrying a non-functional
                                or non-expressed mecA

                                Resource issues
                                reduce widespread use

Table 5. CLSI-recommended reference MIC and zone
diameter breakpoints for S. aureus (4)

Antimicrobial agent   Disc content
                      ([micro]g) *

Methicillin                5

Oxacillin                  1

Cefoxitin                  30

Vancomycin                 --

Teicoplanin                30

Clindamycin                2

Daptomycin                 --

Linezolid                  30

Rifampin                   5

Quinupristin-              15
dalfopristin

Trimethoprim-          1.25/23.75
sulfamethoxazole

                                Zone diameter breakpoints
                                      (nearest mm) *
Antimicrobial agent
                              S             I           R

Methicillin           [greater than or    10-13   [less than or
                      equal to] 14                equal to] 9

Oxacillin             [greater than or    11-12   [less than or
                      equal to] 13                equal to] 10

Cefoxitin             [greater than or     --     [less than or
                      equal to] 22                equal to] 21

Vancomycin                   --            --           --

Teicoplanin           [greater than or    11-13   [less than or
                      equal to] 14                equal to] 10

Clindamycin           [greater than or    15-20   [less than or
                      equal to] 21                equal to] 14

Daptomycin                   --            --           --

Linezolid             [greater than or     --           --
                      equal to] 21

Rifampin              [greater than or    17-19   [less than or
                      equal to] 20                equal to] 16

Quinupristin-         [greater than or    16-18   [less than or
dalfopristin          equal to] 19                equal to] 15

Trimethoprim-         [greater than or    11-15   [less than or
sulfamethoxazole      equal to] 16                equal to] 10

                                    MIC standard ([micro]g/mL) *
Antimicrobial agent
                            S           I            R

Methicillin           [less than or    --    [greater than or
                      equal to] 8            equal to] 16

Oxacillin             [less than or    --    [greater than or
                      equal to] 2            equal to] 4

Cefoxitin             [less than or    --    [greater than or
                      equal to] 4            equal to] 8

Vancomycin            [less than or    4-8   [greater than or
                      equal to] 2            equal to] 16

Teicoplanin           [less than or    16    [greater than or
                      equal to] 8            equal to] 32

Clindamycin           [less than or    1-2   [greater than or
                      equal to] 0.5          equal to] 4

Daptomycin            [less than or    --           --
                      equal to] 1

Linezolid             [less than or    --           --
                      equal to] 4

Rifampin              [less than or     2    [greater than or
                      equal to] 1            equal to] 4

Quinupristin-         [less than or     2    [greater than or
dalfopristin          equal to] 1            equal to] 4

Trimethoprim-         [less than or    --    [greater than or
sulfamethoxazole      equal to] 2/38         equal to] 4/76

S-Susceptible; I-Intermediate resistance; R-Resistant

* Reproduced, with permission, from CLSI publication
M100-S19, Performance Standards for Antimicrobial
Susceptibility Testing, Table 2C. Copies of the
current edition may be obtained from Clinical
and Laboratory Standards Institute, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania
19087-1898, USA. www.clsi.org

Table 6. VISA and YRSA detection

                        Suitable for:

Test                 VISA         VRSA

Screen test          Yes           Yes

Broth dilution       Yes           Yes

Agar dilution        Yes           Yes

Etest                Yes           Yes

Diffusion method      No    Yes (but results
                            may be equivocal)

Automated methods     No           No
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Author:Zurita, Jeannete; Mejia, Carlos; Guzman-Blanco, Manuel
Publication:The Brazilian Journal of Infectious Diseases
Article Type:Report
Geographic Code:0LATI
Date:Nov 1, 2010
Words:6786
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