The Epidemiology Of MRSA Infections Biology Essay


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Not long after the introduction of methicillin into clinical use, the occurance of methicillin resistant Staphylococcus aureus (MRSA) began to escalate in hospitals. (Malhotra-Kumar et al. 2008).Recently however, the epidemiology of MRSA infections has changed globally. From being strictly nosocomial, MRSA has now emerged as a community - associated pathogen (CA-MRSA) (Larsen et al. 2009)

The routine screening procedure, used in the Microbiology laboratory, Galway University Hospital, (GUH), takes at least 72 h to obtain a definitive result. Since detection of MRSA is essential to optimize treatment administration time; minimize patients' discomfort and to reduce costs, a medium that promises rapid and reliable detection of MRSA within 20 hours would increase detection and diagnosis significantly and thus aid in increasing antimicrobic administration, and ultimately lead to a decrease in the MRSA epidemic (Perry et al. 2004)A novel chromogenic medium for the detection of methicillin-resistant Staphylococcus aureus (MRSA), Oxoid brilliance MRSA agar, promised such advantages, (OXOID LTD, 2008) (PERRY, J D et al., 2004) and was thus, evaluated with clinical samples in the Microbiology Laboratory, Galway University Hospital, Galway, Ireland (GUH).

A total of 325 clinical swabs were screened for MRSA on both agars. The sequence of inoculation was randomized to reduce bias. The use of latex agglutination kits and antibiotic susceptibility testing allowed for the confirmation of suspected MRSA isolates. The plates were read as per manufacturers' recommendations:

Biomérieux plates post 24 and 48 hours aerobic incubation (36+/- 1°C)

Oxoid plates post 18-20 hours aerobic incubation (36+/- 1°C)

In total 43 MRSA strains were isolated 27 of which were detected by the Oxoid Brilliance agar and 43 were detected by the routine screening method.

Sensitivity was resulted as 67.5% and 100%, and Specificity was 96.9% and 98.9% for Oxoid and Biomérieux media respectively.

The conclusion of the study would suggest that due to its superior sensitivity for MRSA and the fact that less confirmatory testing was required- thus lowering costs and increasing detection time of confirmed isolates, the recommendation would be for the continued use of biomérieux chromID agar in the routine screening of MRSA.

Glossary of Terms

MRSA Methicillin Resistant Staphylococcus aureus

CLED Cysteine Lactose Electrolyte Deficient agar

MH Muller Hinton agar

AST Antibiotic Susceptibility Testing

PPE Personal Protective Equipment

PPV Positive Predictive Value

NPV Negative Predictive Value

CLSI Clinical Laboratory Standards Institute

VRE Vancomycin Resistant Enterococci

PP Purity Plate

FOX Cefoxitin

PMRSA Presumptive MRSA

CMRSA Confirmed MRSA

ATCC American Type Culture Collection

Table of contents

1.0 Introduction

1.1 Staphylococcus aureus

staphylococcus aureus

Figure Staphylococcus gram stain (X100)(Kimicontrol 2010)

Staphylococci (genus Staphylococcus),are round, gram positive cocci which can divide in any plane and tend to be seen as distinctive grapelike clusters upon gram staining (Figure1). They are non motile; non flagellate and non spore forming. Staphylococci are facultative anaerobic but grow best aerobically. The ability of Staphylococcus aureus (S.aureus) to produce coagulase distinguishes it from other Staphylococcus strains. (Ryan 1994) (Nahimana, Francoli & Blanc 2006) Although S. aureus is part of the normal flora of healthy individuals and is carried in the anterior nares of approximately 20-30% of humans (Meehan et al. 2009)-refer to Table1, strains such as MRSA have the potential to cause disease. (Lelievere, Lina & Jones 1999) (Currie et al. 2008)

Table Features of human Staphylococci






*S. aureus


Anterior nares, perinium





Anterior nares, skin





Urinary tract



1.2 Emergence of MRSA

When penicillin first came into use, many S.aureus strains were susceptible to its affects. However, now, mainly due to its over prescription as an antimicrobic S.aureus emerged as resistant over time due to penicillinase production of some strains.

1.3 MRSA- Mechanisms of bacterial resistance

Resistance tends to be acquired by transduction or via conjugation between strains of S.aureus. Some multi-resistant strains have developed resistance to penicillin's and cephalosporin's and therefore led to epidemics in community and especially nosocomial infections almost globally (Chambers & De Leo September 2009) These strains are commonly referred to as methicillin resistant S.aureus, though they are resistant to many beta-lactam antibiotics. (Gold 1996) (Sevin, Larmaraud-Sevin & Legrand 1999)The main mechanism by which MRSA resistance is achieved is down to chromosomal attainment of the mec A gene. This gene codes for a new peptide - penicillin binding peptide PBP-2 - that has low affinity for β-lactams. The PBP-2 peptide is capable of replacing other staphylococci transpeptidases in cell wall synthesis in the presence of β-lactams, thereby establishing resistance. (Chambers & De Leo September 2009) Figures2 and 3 appendices

'Hospitals in Galway, Dublin and Cork had the highest number of MRSA infections last year despite an overall fall in patients contracting the superbug'. (O'Regan 2009, Tuesday July 14) (O'Regan Tuesday October 13 2009)

'Over 7,500 patients were surveyed in 44 acute Irish hospitals with the overall figure of MRSA infection emerging as under 0.5% -Irish hospitals recorded an overall figure of 4.9%' (Health Service Executive, Ireland (HSE) 2007)

A recent study identified 12% of MRSA isolates as being community-associated, and skin and soft tissue infections were more common among community-associated cases compared with those acquired in hospital or in healthcare associated institutions (Naimi TS Le Dell KH 2003)

1.3 The principle behind Chromogenic agars

Since the first report of methicillin-resistant Staphylococcus aureus (MRSA) as a major nosocomial pathogen in the 1960s, the incidence of infections due to this organism has continued to rise (Gold 1996) (Klevens October 17, 2007). Infections caused by MRSA increase the length of hospital stays and are responsible for rising health care costs, and have a high mortality rate (Carbon 1999) (Jonathan R. Edwards 2009)

In the screening of patients who are at risk to MRSA infection, chromogenic medium, selective for MRSA must be sensitive and capable of detecting suspect colonies efficiently. These agars usually consist of many elements that allow for the growth of only the desired microorganism, i.e. MRSA. These selective mediums inhibit most other bacterial growth and where growth is not inhibited, colonies that form will yield a different colour or no colour at all to allow for the differentiation. (Ryan 1994)

1.4 The chromogenic media used for the detection of MRSA isolates

1.4.1 BIOMÉRIEUX chromIDâ„¢ MRSA Agar

This is a clear medium that bases the isolation of MRSA upon both its resistance to cefoxitin, which is included in the media and chromagen that detects the α-glucosidase activity, which gives rise to the distinctive green colonies. Inhibition of other bacterial growth and the differentiation between MRSA and other Staphylococcal species; that can yield white; pink and/or mauve colonies leads to the increased reliability; sensitivity and specificity of this medium in the screening of MRSA. (Biomerieux 2009)


This is an opaque medium that consists of chromagen that yields denim blue colonies due to the phosphatase activity of many Staphylococci species. To enable differentiation between these strains and MRSA, a marker (other than cefoxitin) is also included, which suppresses the phosphatase enzyme of all other Staphylococci apart from MRSA.This then allows for the isolation of only MRSA present to produce denim blue colonies thereby, increasing the sensitivity and specificity of the media for the detection of MRSA. (Oxoid Ltd 2008)

1.4 Objectives

The aim of this study was to evaluate the ability of a new MRSA chromogenic agar; Oxoid Brilliance MRSA agar, for the detection of MRSA in clinical swabs post 18h incubation compared to that of the agar currently in use; bioMérieux chromID MRSA agar, after 18h and a further 24h re-incubation, at the request of the Microbiology Department at GUH. bioMérieux chromIDâ„¢ MRSA agar and Oxoid Brillianceâ„¢ agar are selective and differential chromogenic mediums for the qualitative detection of methicillin resistant Staphylococcus aureus (MRSA) colonization. The test was performed using swab specimens to screen for MRSA colonization. Monitoring and detection of MRSA infections in this manner may aid in the surveillance and reduction of MRSA outbreaks. (Perry, Rennison & Butterworth Oct. 2004), ( (Nahimana, Francoli & Blanc 2006) (Muto, Jernigan & Ostrowsky 2003) (Sevin, Larmaraud-Sevin & Legrand 1999)

The comparison was based upon their sensitivity; specificity; ease of use; cost effectiveness and rapidity in obtaining reliable confirmatory results

2.0 Materials and Methods

2.1 Materials used

2.1.1 Media used

BioMérieux MRSA ID chromagar plates

Oxoid Brilliance MRSA chromagar plates

CLED agar plates

Muller Hinton agar plates

2.1.2 Antimicrobial Susceptibility Testing (AST)

The materials used were;

Cefoxitin discs (30µg) (Oxoid)

Oxoid Disc Dispenser (Cefoxitin 30)

Vortex genie 2â„¢ (BioMérieux)

Vitek densiChekâ„¢ (BioMérieux)

ATCC control strains

Saline (5ml sterile tubes )

2.1.3 REAGENTS used

Pastorexâ„¢ Staph-plus latex reagent kit

Sterile swabs

Sterile 1µl loops

Plate racks

Test-tube racks

4°C cold room

36°C +/- 1°C O² incubator

BioMérieux ID-ASEâ„¢ Catalase reagent

MRSA clinical specimen swabs (received daily)- in charcoal transport medium

2.2 Methods of screening for MRSA

2.2.1 Specimen collection and transport

The sterile swab is the most convenient method of specimen collection for MRSA screening.. (Ryan 1994), therefore the use of swabs within transport tubes containing charcoal was ideal, since they are designed to maintain the viability of any bacteria present without significant increase in growth due to being non nutritive. (Aimes 1967)

2.2.2 Specimens used

A total of 325 swabs were used in this trial-sourced from GP and hospital patients (December 2009-January 2010). The most common swabs used for MRSA screens tended to be:



Groin and


Table 4.1 Sensitivities of Body Sites for the Detection of MRSA- Results

Graph4.2 Percentage of MRSA isolation based on swab site- Results

'Active surveillance for patients colonized with methicillin resistant Staphylococcus aureus (MRSA) is recommended to prevent MRSA infections in health care settings. The sensitivity of patient screening for MRSA colonization is partially dependent on the body sites sampled. The nose and open skin areas (i.e., wounds and device exit sites) are considered the most important sites for colonization However, studies have suggested that a substantial proportion of colonized patients will be missed if only these anatomic sites are sampled' (Journal of Clinical Microbiology 2008)

2.2.3 Good Laboratory Practice

All specimens received were considered as infectious.

Asceptic technique and normal precautions were observed while handling all specimens, reagents and microbial cultures throughout the evaluation.

2.3 Culture methods

2.3.1 Direct Inoculation

Each swab was directly inoculated onto both MRSA selective agars; streaking for discrete colonies was not required (Figure 4-appendices). The sequence of inoculation was randomized to reduce bias (Haekel, Rainer and Wosniok, Werner, 2007).

Day 1: BioMérieux MRSA agar was swabbed first then Oxoid Brilliance MRSA agar.

Day 2: Oxoid Brilliance MRSA agar was swabbed first then BioMérieux MRSA agar. The alternation of inoculation continued as such for each day of the trial, until all Oxoid plates were used.

Plates were inoculated directly on the day of receipt of the swab¹, incubated at 36°C aerobically, and read after 20 and 48 h (18-20 h incubation recommended for Oxoid media. (Oxoid Ltd 2008)

¹Prior to media inoculation, all plates used were allowed to reach room temperature. Any unused plates were re-fridgerated (4°C).

Figure 4 Direct inoculation methods - appendices

2.4 Identification and confirmation of MRSA isolates

Colonies suggestive of MRSA were confirmed as Staphylococcus aureus by the use of latex agglutination kits and antibiotic susceptibility testing (AST) for MRSA confirmation .

The plates were read as per manufacturers' recommendations:

BioMérieux plates post 20 and 48 hours aerobic incubation (36+/- 1°C)

Oxoid plates post 20 hours aerobic incubation (36+/- 1°C)

Purity plates were set up on CLED agar where low level colonization occurred (<10 colonies) and/or if growth was mixed.

Refer to appendices:

Figure 5a Setting up purity plates on CLED agar

Figure 5b S. aureus colonies (yellow) on CLED - coagulase positive

Figure 6 AST methods

Refer to results

Table 4.5 AST results for PMRSA colonies on Oxoid media

2.4.1 Latex Agglutination

Staph Latex - PASTOREX â„¢ - BIO-RAD - Test principle

The test is designed to allow for the differentiation of S.aureus from coagulase negative Staphylococci (CNS). The combination of fibrinogen, IgG and anti-capsular monoclonal antibodies and protein A ('clumping factor')allows for the recognition of S.aureus by the agglutination which forms upon emulsifying a few suspect colonies with the reagent mixture. This agglutination is visible macroscopically within a few seconds approximately. See Diagram 1.

A positive result was visible agglutination with the latex reagent.

No agglutination occurred with the negative control thus ensuring results were valid

(BIO-RAD 2010)

Diagram 1 Latex agglutination methods

2.4.2 Antimicrobial Susceptibility Testing Cefoxitin Disk Diffusion

Distinguishing presumptive MRSA isolates was an essential part in determining whether true MRSA's were isolated.

For MRSA, the CLSI recommend the use of 30µg cefoxitin disk on Mûller Hinton agar with 0.5 Mc Farland suspension inoculums, using sterile swabs. (Swenson 2005) (Clinical and Laboratory Standards Institute 2003)After incubation for 24h, the zone diameters were measured in mm, and compared to published figures for interpretation. Zone sizes ≤21mm confirmed MRSA isolation (table 4). (Clinical and Laboratory Standards Institute 2005)

All colonies showing resistance to Cefoxitin were re-tested for the presence of clumping factor. (Colonies tested were taken from directly around the zone diameter).

The Clinical and Laboratory Standards Institute (CLSI) recommends the Cefoxitin disk screen test, the latex agglutination test for PBP2a, alternative methods of testing for MRSA (Clinical and Laboratory Standards Institute 2005)

Table 2 CLSI published figures of zone diameter limitation in the confirmation of MRSA

*Susceptible implies that the organism is readily inhibited by the antimicrobic

**Resistant implies that the organism is not inhibited by the antimicrobic

2.5 Quality Control.

As a validation procedure, Staphylococcus aureus ATCC® 43300 *MRSA positive and Staphylococcus aureus ATCC® 25923 *MRSA negative were used as daily Internal quality control (IQC) strains- refer to Table 3a. These were the only ATCC strains available for use at the time of this trial; see Tables 3b and 3c for both manufacturers' recommendations.






S. aureus

ATCC 43300*

Blue colonies

Green colonies

S. aureus

ATCC 25923*

Recommended ATCC strains for use with bioMérieux chromIDâ„¢ MRSA agar

S. aureus ATCC® 29213 - mecA negative Expected Result: Inhibition

S. aureus ATCC® 25923 - mecA negative Expected Result: Inhibition

S. aureus ATCC® 43300 - mecA positive Expected Results : Green colonies

Table 3b (Biomerieux 2009)

Recommended ATCC strains for use with Oxoid Brillianceâ„¢ MRSA agar

Staphylococcus aureus NCTC10442 Expected results: Blue colonies

Staphylococcus aureus ATCC®33591 Expected results: Blue colonies

Staphylococcus epidermidis ATCC®12228 Expected results: Inhibited

Staphylococcus aureus ATCC®25923 Expected results: Inhibited

Table 3c (Oxoid Ltd. 2008)

3.0 How should Clinical Laboratories screen for MRSA?

Laboratory screening for MRSA lends itself to being a complex balance between;




Speed of reporting results

Both Oxoid Brillianceâ„¢ and bioMérieux chromIDâ„¢,are chromogenic media that combine primary growth factors among others, which claim to enhance their ability and sensitivity to differentiate MRSA from CNS.

3.1 The chromogenic media used in the identification of MRSA isolates

Oxoidâ„¢ brilliance MRSA chromagar

MRSA appeared as denim blue / navy* colonies on an opaque medium

Non MRSA appeared as either no growth (inhibition) or white colonies

bioMêriuxâ„¢ MRSA ID chromagar

MRSA appeared as green colonies* on a clear medium

Non MRSA appeared as either no growth (inhibition) or colourless colonies

3.2 Media Interpretation

OXOIDImage 1 Actual images of MRSA ATCC 43300 on both media



Image taken 08 February 2010

4.0 Results

4.1 Result reporting protocol

For the purpose of this study the result reporting protocol was as follows:

If presumptive MRSA (PMRSA) colonies grew on a medium and were later confirmed as MRSA (CMRSA) - the result was considered a true positive.

When a sample was found to be negative for MRSA, on both media, the result was assumed as a true negative.

If MRSA - like colonies were yielded, but were not confirmed via further testing, the result was deemed to be a false positive.

An isolate that was confirmed as MRSA on one media, but failed to grow on the other, was resulted as a false negative.

If either medium failed to allow growth of MRSA when the other medium did, this was resulted as a false negative.

4.2 Results obtained

The results obtained for both media are summarized below and in Table 4.1 for

bioMérieux chromID and Oxoid Brilliance respectively. (n=336)

Oxoid Brilliance gave yields of;

9 false positives

27 true positives

13 false negatives

287 true negatives

bioMérieux chromID yielded;

3 false positives

43 true positives

0 false negatives

290 true negatives

Table 4.1- Results for bioMérieux chromIDâ„¢ media (48h) and Oxoid Brillianceâ„¢ media (20h)



bioMérieux chromID


Oxoid Brilliance







Test Positive






Test Negative












4.3 Data analysis

From the results obtained, (Table 4.1), the respective sensitivities; specificities; positive and negative predictive values (PPV AND NPV) for both media were calculated as follows;

The SPECIFICITY of a test is the probability that a test will produce a true negative result when used on a non-infected person/specimen.


287/287+9 = 96.9% Oxoid

290/290+3 = 98.9% BioMérieux

The SENSITIVITY of a test is the probability that it will produce a true positive result when used on an infected person/specimen


27/27+13 = 67.5% Oxoid

43/43+0 =100% BioMérieux

The POSITIVE PREDICTIVE VALUE of a test is the probability that a person is infected when a positive test result is observed.


27/27+9 = 75% Oxoid

43/43+3 = 93.4% BioMérieux

TP - true positive FP - false positive TN - true negative FN - false negative

These calculated figures are summarized overleaf as Table 4.2 and by Graph 4.3

Table 4.2 Results for both media evaluated in relation to their sensitivity; specificity; positive and negative predictive values (PPV/NPV)















Graph 4.3

Graphical representation of results for both media evaluated

It can be seen from these results both media have similar specificities for MRSA, with bioMérieux slightly outperforming Oxoid in this area. However, chromID showed a substantially better performance with regards to sensitivity. Even after 20h incubation, a detection rate of approximately 90% for MRSA, isolated as green colonies on chromIDâ„¢ was observed, this increased to 100% post 48h incubation, giving a sensitivity of 100% for bioMérieux chromIdâ„¢. In Comparison, a 62.7% detection rate and 67.5% calculated sensitivity was resulted for Oxoid Brillianceâ„¢. This is in contrast to previously reported figures of 99.2% (Oxoid Ltd. 2008) for sensitivity of this medium. bioMérieux chromIdâ„¢ proved far superior in regards to its sensitivity for the detection of MRSA.

4.4 Distribution of MRSA isolates based on specimen swab site

The main human habitat of MRSA tends to be the anterior nares and skin. These results demonstrate again bioMérieux chromID as being more sensitive at detecting MRSA colonization than Oxoid Brilliance due to its ability to not only detect MRSA from sites that are predisposed to increased MRSA presence, but also of those sites that may contain a low level of colonization. (Graph 4.4 ; 4.5 &Table 4.5)

Graph 4.4 Percentage of MRSA isolates based on specimen swab site type (n=43)



Other *





% Specimens Received




True Positives




% MRSA Carriage




Table 4.5

Sensitivities of Body Sites for the Detection of MRSA among Colonized Patients Identified By Culture Based Screening during the Comparison Study (December 2009-January 2010) (*Other swab sites included - Ankle; wound; groin; ulcer and catheter site).

Graph 4.5 percentage yield of MRSA on both media

Specimen ID

Zone diameter to cefoxitin(mm)

Latex reaction


























































4.5 Results for AST

Table 4.6 Confirmation of MRSA isolates on Oxoid Brillianceâ„¢

Where low level or mixed growth occurred of PMRSA colonies, AST was performed to confirm each isolate as MRSA

4.5.2 AST results for false negatives that grew on Oxoid Brillianceâ„¢

Table 4.7 Recheck of false negatives on Oxoid Brilliance â„¢ which were true positives on bioMériux chromIDâ„¢.

Specimen ID

Growth observerd

Swab site

Latex reaction





















A number of false negatives observed for Oxoid Brilliance yielded growth of white; pink and mauve colonies. These were latex and AST tested, and subsequently confirmed as MRSA (refer to Table 4.7). This suggests that Oxoids medium is either not sensitive enough to the phosphatase activity of MRSA or that there is a delay in its production by the microorganism. Either way, this only reinforces the inadequacies of the medium for MRSA detection.

*K66 AND 69 only became positive (blue colonies) at greater than 48h incubation.Plates were inoculated - 04/01/2010, and did not yield blue colonies until - 06/01/2010

All the above were positive on bioMérieux chromIDâ„¢ (48h incubation)

4.6 ATCC controls tested on both media

Table 4.8 Results for ATCC strains tested on both media

Control strain

Oxoid Brillianceâ„¢

bioMérieux chromIDâ„¢




Staph. aureus MRSA negative ATCC 29213



Staph.aureus MRSA positive ATCC 43300



Staph. aureus MRSA neg. ATCC 976



Staph. sciuri ATCC 29061



Staph. saprophyticus ATCC 15305



Staph.epidermidis ATCC 12228



Staph. aureus



Staph. aureus ATCC 977



Enterococcus faecalis ATCC 51299 VRE positive



Enterococcus faecalis ATCC 29212 VRE negative *



* See Image 2

*Growth indicates denim blue / green colonies grew on the agar. (Oxoid/Biomérieux respectively).

*No growth indicates either inhibition of growth or growth of non - PMRSA colonies(Latex negative CN

The lack of specificity by Oxoid Brillianceâ„¢ MRSA agar was evident here by the growth of numerous ATCC control strains that were non- MRSA. Each non- MRSA strains that grew on Oxoids medium yielded blue colonies, identical to that expected for MRSA isolates. bioMérieux chromIdâ„¢ MRSA agar, did allow the growth of one non-MRSA, ATCC control strain, however colonial morphology and colour differed from that expected of an MRSA isolate, thus allowing for differentiation between significant and non significant growth.(see Image 2 ) This reduced the need for unnecessary further testing, thereby reducing costs and increased rapidity of reporting results.

Image 2 False positive growths on both media

Colonies easily distinguishable

Colonies undistinguishable

VRE growth on Oxoid media VRE growth on Biomérieux media E:\100V1003\100_0004.JPG

Growth on both media of Enterococci -VRE negative ATCC 29212 strainE:\100V1003\100_0001.JPG

BioMérieux chromIDâ„¢ did allow the growth of one non-MRSA control strain, however the growth was very slight compared to the growth on Oxoid Brillianceâ„¢. There was also a definite difference in colonial appearance and colour produced, (smaller and paler green colonies for the VRE negative strain than the Larger, more vivid green colonies of MRSA). [Image taken 08/02/2010]

The results obtained for sensitivity and specificity for both Oxoid Brillianceâ„¢ and bioMerieux chromIDâ„¢ were used to assess their theoretical predictive values (probability for TP; FP; FN and TN),

4.7 Hypothetical assessment of both media for routine use

Table 4.8 Estimated predictive values for both media with respect to MRSA detection

bbioMérieux chromIDâ„¢







probability that it will be positive


probability that it will be negative


For any positive test result:

probability that it is a true positive

["positive predictive value"]PPV

probability that it is a false positive

For any negative test result:

probability that it is a true negative

["negative predictive value"]NPV

probability that it is a false negative

Oxoid Brillianceâ„¢Bottom of Form







probability that it will be positive


probability that it will be negative


For any positive test result:

probability that it is a true positive

["positive predictive value"]PPV


probability that it is a false positive


For any negative test result:

probability that it is a true negative

["negative predictive value"]NPV


probability that it is a false negative


Using the results obtained for sensitivity and specificity for both media evaluated, the theoretical predictive values were estimated .The results in Table 4.8 were estimated, given that the *prevalence of MRSA within the Republic of Ireland is 0.049 (4.9%), (Health Service Executive, Ireland (HSE) 2007)

The estimated results above demonstrate that should Oxoid Brillianceâ„¢ agar, replace bioMérieux chromID, as the routine method of culture screening for MRSA, the detection rate would be significantly reduced (83.7% versus 52.8% for bioMérieux and Oxoid respectively- see above Table 4.8).

This would also lead to a drop in treatment administration and ultimately an increase in HA-MRSA and CA-MRSA infections.

5.0 Discussion

MRSA is a serious public health problem which exerts huge pressure on the healthcare system. A strategy used to combat this is admission screening using several MRSA swabs taken from mucocutaneous colonization sites of high-risk patients (MRSA screening). It has also been reported that the speed with which MRSA carriage is detected has an important role to play as a key component of any effective scheme to prevent the pathogen from spreading. (Sturenburg & LADR GmbH 2009) Rapid reporting of screening results will improve MRSA control, provided that a clear action plan for positive cases is in place and is being followed. (SARI infection control subcommittee 2008)

An effective culture screening method is direct inoculation of specimen swabs on a well-performing MRSA-selective chromogenic agar. This method usually produces a positive result after 24 h of incubation in >95% of true-positive cases (Diederen 2006) and is sufficient for most initial treatment and infection control decisions. Full antimicrobial susceptibilities, by the Cefoxitin disk diffusion method, can then be performed the next day (48h agar)to confirm MRSA isolation. (Bannerman 2003)

Since current MRSA culturing methods involve a 2-3 day delay before the final results are available, rapid culturing media, such as Oxoid Brillianceâ„¢ MRSA agar, have been developed and should they prove reliable, their implementation would reduce the time of detection of MRSA carriers from 48-72 to 18-20.

This study took into account all aspects of an effective media for the detection of MRSA from clinical specimen swabs, basing the results obtained upon sensitivity; specificity; PPV; NPV and economic viability to determine the best media that would suit this purpose.

5.1 Sensitivity

Post 24h incubation, nasal positive specimens yielded green colonies on bioMérieux chromIDâ„¢ agar, most swabs from other sites became positive after 48h incubation - this was most likely due to the increased presence of MRSA in anterior nares than other body sites, thus, they require further incubation to produce significant growth for detection-, therefore, the 48h incubation with BioMérieux plates seems to be justified. Although Oxoid Brillianceâ„¢ MRSA media does not require further incubation, their plates frequently showed MRSA isolates, previously confirmed as positive on BioMérieux media as negative.

There was also a marked increase in false positives on Oxoid Brillianceâ„¢ agar in comparison to bioMérieux chromIdâ„¢ upon incubation for a further 24hrs. An inoculum effect may have explained the differences in the sensitivities obtained for both media tested, since one swab was used to inoculate both selective agars. However, this would have affected all results equally. Since swabs were inoculated in a manner to reduce such bias, Oxoid Brillianceâ„¢ MRSA lack of sensitivity during this trial was constant, with no increase or decrease in its sensitivity for MRSA detection based upon the sequence of inoculation. MRSA was isolated from 43 (12.7%) of the 336 specimens that were screened. BioMérieux chromIDâ„¢ detected 43 (100%) MRSA isolates compared to only 27 (67.5%) MRSA isolates, that grew on Oxoid Brillianceâ„¢. (Table 4.1 Results)

Of the 43 MRSA isolates recovered on bioMérieux chromIDâ„¢ agar, >90% grew after overnight (20h) incubation.Oxoid Brillianceâ„¢ failed to recover MRSA from 13 MRSA confirmed specimens.

5.2 Disparity /Inconsistency

BioMérieux agar allows for the differentiation between MRSA and non-MRSA colonies that may both give rise to green colonies by the difference in colonial morphology and degree of the green colour. Oxoid's agar, on the other hand, provided no distinguishing factors that allowed for the differentiation between MRSA and non-MRSA isolates see figure 9-11 appendices.

Distinguishing between colonies that yielded blue / green colonies on each medium, but may not have been MRSA, was another factor in reducing the cost of further testing and decreasing the turnaround time (TAT) of result reporting/authorization.

Small pale green colonies, mainly after 48h incubation on bioMérieux agar were easily distinguishable from MRSA strains-large vivid green colonies, but no distinctive factors were evident with Oxoid's media, with non - MRSA colonies exhibiting comparable colonial morphology and colour to MRSA.

Another issue encountered with Oxoid's medium was that many colonies, though not producing the denim blue colour, were to be later confirmed as MRSA on bioMérieux chromIDâ„¢ (at 20/48hr). Many, ultimately were positive for Oxoid Brillianceâ„¢ , but at >48hr, alerting to the fact that the media was not performing as desired and not as sensitive to MRSA detection as promised with a sensitivity of 67.5% compared to 100% for BioMérieux.(Table 4.5.2 Results)

5.3 Ease of use

At GUH, swabs for inoculation were allocated one half of each plate.. It was found that since BioMérieux agar is a clear medium, it was easy to see where to inoculate, whereas, with Oxoid Brillianceâ„¢, being an opaque medium, it was not. This could lead to cross contamination between swabs inoculated and thus lead to an increase in false positives. This may seem trivial, but since the volume of specimens received daily was substantial, it would certainly be more cost effective not to allocate a single pate for each patient swab

5.4 Cost effectiveness

Although, theoretically, Oxoid's agar would be more cost effective - €0.80c versus €1.04 Biomérieux per plate - in reality this would not be the case, since, as observed in this trial, the tendency of the agar to allow growth of microorganisms other than MRSA (yielding blue colonies) leads to unnecessary time consuming and costly further confirmatory testing.

5.5 Findings

The yields of MRSA from various anatomical sites were dissimilar for the two media. From a total of 325 swabs and 11 ATCC strains, inoculated on both media, bioMérieux chromIDâ„¢ detected 43 MRSA isolates, Oxoid Brillianceâ„¢ detected only 27.

The number of specimens with suspicious colonies requiring further investigation which turned out not to be MRSA (false positives) differed substantially between the two media, with 9 of 336 (2.6%) for Oxoid Brillianceâ„¢ compared to only 3 of 336 (89%) for bioMérieux chromIDâ„¢ .

Senitivity was resulted as 67.5% and 100%, and Specificity was 96.9% and 98.9% for Oxoid Brillianceâ„¢ and bioMerieux chromIDâ„¢ respectively.

Taking into account all of the above it seems that sensitivity and specificity are in all likelihood the most valuable of the indicators in the comparison between both agars for MRSA isolation. Biomerieux chromIDâ„¢ agar was found to be easier to interpret, more specific and had better sensitivity than Oxoid Brillianceâ„¢ for MRSA detection.

6.0 Conclusion

Due to the importance of MRSA screening in a time of increased nosocomial and community acquired MRSA infections, the use of a media that is sensitive in its ability to detect MRSA infection, is vital in the rapid administration of treatment and thus, the decrease in its spread.

The tendency of Oxoid's Brillianceâ„¢ medium to facilitate growth of non MRSA isolates, with little or no differentiation to true MRSA isolates, and more significantly, the number of confirmed MRSA isolates it failed it identify during this trial, makes this medium unsatisfactory in respect to its use as an MRSA selective chromogenic agar.

For these reasons, although Oxoids Brillianceâ„¢ agar should, theoretically, detect MRSA earlier then the media currently in use, its lack of specificity and more so sensitivity were not advantageous as an alternative screening tool for the detection of MRSA.

In comparison, BioMérieux chromID medium was easy to interpret; had a high sensitivity even after 24h (>90%) and required less confirmatory testing, consequently reducing costs and TAT in result authorization.

Consequently, I would recommend the continued use of Biomerieux chromIDâ„¢ MRSA agar, for culture based MRSA screening.

7.0 Appendices

Waves of resistance: Staphylococcus aureus in the antibiotic era

Figure 2

mecA gene attainment - MRSA mechanism of resistance

The main mechanism by which MRSA resistance is achieved is down to chromosomal attainment of the mec A gene. This gene codes for a new peptide - penicillin binding peptide PBP-2 - that has low affinity for β-lactams. The PBP-2 peptide is capable of replacing other staphylococci transpeptidases in cell wall synthesis in the presence of β-lactams, thereby establishing resistance. (Chambers & De Leo September 2009)

Figure 3 S.aureus mechanisms of resistance (Nizet et al 2001)

Figure 4 Direct Inoculation Method

Any new specimens received in the afternoon, were swabbed directly onto both plates in the same order designated for that day, ensuring each swab was re-immersed in transport medium before inoculation of the proceeding plate. (Microbiology Dept.,Galway University Hospital 2009)

Where mixed or scanty growth occurred, purity plates were set up on CLED agar and incubated for 24hrs, at 37°C.

These colonies, (yellow on CLED), were confirmed as latex positive before a Cefoxitin disk diffusion test was done. (Microbiology Dept.,Galway University Hospital 2009)Figure 5a - Setting up Purity Plates

Figure 5b - S. aureus colonies (yellow) on CLED - Latex positive


Figure 6: Procedure for AST

CLSI figures were developed by zone comparisons; MIC; blood level data and from studies on zone diameter distributions (susceptibilities), of numerous species of micro-organisms of known sensitivity/resistance.

For MRSA, CLSI recommend the use of 30µg Cefoxitin disk on Mûller Hinton agar with 0.5 Mc Farland suspension inoculums, using sterile swabs. (Clinical and Laboratory Standards Institute 2005)

Figure 7

Figure 8

Figure 9

MRSA and non- MRSA growth on Oxoid Brillianceâ„¢ Medium

E. Faecalis 29212 on Oxoid medium



Enterococcus Faecalis ATCC 29212 ISOLATED ON OXOID MEDIAE:\100V1003\100_0001.JPGE:\methodology\agar pics\DSC00719 (1).JPGC:\Documents and Settings\admin\Local Settings\Temporary Internet Files\Content.Word\100_0005.jpgC:\Documents and Settings\admin\Local Settings\Temporary Internet Files\Content.Word\100_0008.jpg

Figure 10 - MRSA growth on both media

E:\methodology\agar pics\DSC00719 (1).JPG

C:\Documents and Settings\admin\My Documents\100V1003\100_0002.JPG

Figure 11- MRSA growth on Oxoid Brillianceâ„¢

Note the difference in colonial morphology between the MRSA isolated, and the presence of white colonies.

the white colonies were later confirmed as MRSA by latex testing and AST>

Both MRSA isolates

Figure 13- MRSA growth on Oxoid Brillianceâ„¢

Note the difference in colonial morphology between the MRSA isolated, and the presence of white colonies.

the white colonies were later confirmed as MRSA by latex testing and AST>

Oxoid Brillianceâ„¢ images of MRSA growth (Oxoid Ltd. 2008)

CNS and VRE growth on Oxoid Brillianceâ„¢ agar

MRSA isolated on Oxoid Brilliance PR023605.jpgChromogenic_MRSA_floating_2 (1).jpgChromogenic_MRSA_floating_2 (1).jpg

These images display the lack of uniformity with regard to MRSA growth on Oxoid Brillianceâ„¢ agar colonial morphology.

Figure 12 - The absence of uniformity observed for MRSA growth on Oxoid Brillianceâ„¢ agar.100_0009.JPGDSC00713 (1).JPG100_0005.JPG100_0008.JPG100_0004.JPGDSC00719 (1).JPG100_0001.JPG5Jan10 Brilliance MRSA 24hrs (1).JPG

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