Comparison of Performance between Mannitol Salt

Agar-supplemented Cefoxitin Disc and Chromogenic Media for

Methicillin-resistance Staphylococcus Aureus Screening

Sri Amelia

, Dian Dwi Wahyuni

, Rina Yunita

and Muhammad Fakhrur Rozi

Department of Microbiology, Universitas Sumatera Utara, Jl. Dr. Mansur No.5,Medan, Indonesia

Faculty of Medicine, Universitas Sumatera Utara, Jl. Dr. Mansur No.5, Medan, Indonesia

Keywords: Methicillin-resistance, Chromogenic.

Abstract: Methicillin-resistance Staphylococcus aureus (MRSA) and its burden still become a major problem in

hospital setting worldwide. Accurate and rapid screening method is needed to prevent transmission We

conducted a study to compare the performance between conventional method, modified-conventional

method, and chromogenic media to detect MRSA carrier. This cross-sectional study was obtained 80 nasal

swabs of medical personnel who worked in Intensive Care Unit. The location of study was in Hasan Sadikin

General Hospital and Sentosa Hospital, Bandung, Indonesia between March and July 2009. Meanwhile,

incubation and identification process were set in Department of Microbiology, Universitas Padjajaran,

Bandung, Indonesia. Modified-conventional method, cefoxitin disc plated at the bottom of Mannitol-Salt

media, was made. The result showed that specificity increased (100%, p-value < 0.001, kappa index= 1.00)

using modified media and similar result was also found with chromogenic media. While using conventional

method alone just produced 98.7% of specificity and 100% of sensitivity (p-value <0.001, kappa

index=0.85). Thus, modified-conventional method can be considerable since its detection rate of MRSA

similarly found as a reliable method for MRSA screening.

1 INTRODUCTION

Since its findings in Detroit in 1981 among injecting

drug users, Methicillin-resistance Staphylococcus

aureus (MRSA) remains problematic. MRSA will

produce low-affinity penicillin-binding protein 2

(PBP2) encoded by mecA gene, and bacteria still

continue its cell-wall synthesis, then it causes using

beta-lactam lineage as antimicrobial therapy is

meaningless (Schroeder et al., 2017). Furthermore,

using highly expensive antibiotics and prolonged

hospitalization of a patients infected-MRSA will

lead to a higher hospital expenditure (Collins, 2010).

MRSA has become emerging problem causing

nosocomial infection, especially in Intensive Care

Unit (ICU), anterior nares is its predilection mostly,

therefore colonization in this structure plays an

important role in MRSA transmission. The

transmission generally involves medical health-

workers who acquired infection and MRSA

colonization in their anterior nares. Transmission

will ensue directly if there is contact with infected-

individual. Endemically, asymptomatic carrier is

presence among hospitalized-patients and commonly

caused MRSA bacteraemia especially in critically-ill

patients (Marcel, 2008). Carrier identification and

isolation (carrier) MRSA are effective method in

controlling its incidence. MRSA screening should be

performed for both inpatients and all medical

personnel working in certain units, especially who

works in Intensive Care Unit (ICU), the screening is

mandatory since it related to morbidity and mortality

of a patients-infected MRSA (Klevens, 2007).

Screening media can be performed using

conventional method, in which the isolates will be

implanted in blood agar then followed by

Staphylococcus aureus identification process up to

susceptibility test against methicillin. In addition to

blood agar, MRSA identification can also use

selective medium for Staphylococcus aureus such as

Mannitol Salt agar (MSA), furthermore, as the

confirmatory test, susceptibility test by using

Muller-Hinton agar (MHA)-supplemented cefoxitin

must be performed. MSA, MHA, and oxacillin

476

Amelia, S., Wahyuni, D., Yunita, R. and Rozi, M.

Comparison of Performance between Mannitol Salt Agar-supplemented Cefoxitin Disc and Chromogenic Media for Methicillin-resistance Staphylococcus Aureus Screening.

DOI: 10.5220/0010075004760480

In Proceedings of the International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches (ICOSTEERR 2018) - Research in Industry 4.0, pages

476-480

ISBN: 978-989-758-449-7

resistant screening agar are commonly used among

other solid conventional methods in Europe.

However, chromogenic media can be used in several

laboratories because it eliminates all the issue related

to time-consuming procedure for MRSA screening

method and it is also widely used in Europe (Fagan,

2010).

As mentioned above, alternative method is

needed for screening MRSA in low-cost laboratory,

high cost and short expired date become a limitation

using chromogenic media as main screening media

for MRSA. Our study was conducted to determine

and compare the performance of conventional

method, modified-conventional method or MSA-

supplemented cefoxitin disc (MSA-CFOX), and

chromogenic media.

2 THE MEDIA

This study involved 80 health-workers who worked

in Intensive Care Unit (ICU) of Hasan Sadikin

General Hospital and Santosa Hospital, Bandung,

Indonesia between March and July 2009. This cross-

sectional study aimed to determine and compare the

performance of modified conventional method and

ChromIDTM for screening MRSA. Firstly, samples

were obtained from nasal swab of all health-workers

who had given their approval. Nasal swabs (BBL

CultureSwab Liquid Stuart) were directly sealed

inside the reaction tube which already contained

transport medium.

Furthermore, the specimens were inoculated into

conventional media (blood agar and Muller-Hinton

agar), modified-conventional media (MSA-

supplemented cefoxitin disc or MSA-CFOX), and

chromogenic media (ChromIDTM MRSA).

Bacterial inoculation and identification processes

were carried out in Microbiology Department,

Faculty of Medicine, Universitas Padjajaran,

Bandung, Indonesia.

2.1 Modified-Conventional Method or

MSA-CFOX

We provided petri dishes with cefoxitin disc placed

at the bottom of the media and it was divided into

three sections. We prepared initially MSA medium

under laboratory roof and directly plated cefoxitin

disc before the media solidified. Finally, modified-

conventional medium or Mannitol-Salt agar

supplemented cefoxitin disc (MSA-CFOX) were

ready to be used and incubated for 24 hours at 37°C.

Previously, we also assessed whether cefoxitin

substance had disseminated all over the

compartment of medium by using High-Performance

Liquid Chromatography (HPLC) method in School

of Pharmacy, Institut Teknologi Bandung (ITB),

Bandung, Indonesia.

After 24 hours, the colonies were seen yellow

and microscopically the bacteria arranged in groups

like grapes, from this simple examination we called

this findings as ‘suspected’ MRSA. For

confirmation, the colonies had also to be inoculated

into Muller-Hinton Agar (MHA) plate by placing

cefoxitin 30 µg disc on it (Kirby Bauer disc

diffusion method). Both medium acquired equal

handling with clearly established procedure. After

24 hours of incubation, observation and

measurement of inhibition zone were performed.

Inhibition zone ≤ 22 mm, it is evident for MRSA

while if ≥ 22 mm is MSSA.

2.2 Conventional Method

Inoculation into blood agar was performed before it

was incubated for 24 hours at 37°C. After

incubation, we obtained colony growth on the

surface of agar. Evaluation of the colony was

performed by microbiologist to determine whether

Staphylococcus aureus colonies are positive or not.

The colonies were circular, smooth, slightly

appeared on surface, glistening, and gray to

yellowish brown. Furthermore, the colony was

obtained using sterile ose and we fixated it on the

object glass for gram staining, catalase test, and

coagulase test. MHA medium was used as a part of

confirmatory for MRSA detection. Previously,

inoculum was incubated in McFarland medium to

increase the number of bacteria. We used cefoxitin

30 µg to determine the inhibition zone. Final results

would determine whether the presence of MRSA is

evident.

2.3 Chromogenic Media

Inoculation was carried out using ChromIDTM

MRSA, France for 24 hours at 370 C. It is well

explained that the colonies would appear greenish

for MRSA colonies.

2.4 Data Analysis and Study Approval

We used fisher exact test for data analysis while data

processing was done by calculating the kappa

coefficient of agreement (K) between the two

methods calculated by 2x2 proportion table. There

are three criteria for the kappa limit value, for

Comparison of Performance between Mannitol Salt Agar-supplemented Cefoxitin Disc and Chromogenic Media for Methicillin-resistance

Staphylococcus Aureus Screening

477

example if the kappa ≥ 0.75 is mentioned as high

conformity, if kappa between 0.40 and 0.75 is called

moderate conformity, whereas ≤ 0.40 is mentioned

as less conformity. Study approval was also obtained

from ethics commission of medical research of

Universitas Padjajaran, Bandung, Indonesia.

3 RESULTS

A total of 80 nasal swabs were examined and

inoculated then. Finally, the results were positive for

MRSA only in four samples (3.8%), and 15 samples

(18.8%) were positive for Methicillin-sensitive

Staphylococcus aureus (MSSA). The rest,

Streptococcus sp. (32.5%), Staphylococcus

saphrophyticus (7.5 %), and Staphylococcus

epidermidis (37.5%) were also found from samples.

After incubation period, we validated three different

methods and compared them with multistep

procedure mentioned above. We presented the result

in Table 1.

Table 1: MRSA identification using three different

methods.

Methods Sensitivity

(

)

Specificity

(

)

value

kappa

Index

Conven-

tional

metho

100

98.7

<.001

0.85

MSA-

CFOX

100 100 <.001 1.00

Isolates that exhibited MRSA positive were four

samples (3.8%), confirmatory test was also done by

procedure mentioned above. Recently, some

molecular methods are introduced to detect MRSA,

detecting mecA gene still become reference tools to

demonstrare methicillin resistance, however,

because of inadequacy of tools and personnel, this

method can hardly be used in most laboratories

particularly in developing countries. Therefore,

proper and inexpensive screening method is still

required for routine procedure (Xu, 2017).

Identification of MRSA is depending on certain

factors, such as cost, speed of result, availability of

tools and equipment, sensitivity, and specificity. For

routine diagnostic purpose, tube coagulase or latex

agglutination, and catalase test should be done at the

beginning of identification process. Afterward,

options for detection methicillin resistance,

including disc diffusion, minimum inhibitory

concentration (MIC) measurements, chromogenic

agar, latex agglutination, rapid screening methods

and molecular detection are the methods can be used

for the next identification process. Media type,

incubation times and temperature are important in

determining the results of methicillin sensitivity. In

addition, by plating before broth enrichment will

increase the sensitivity of the screening method,

particularly using cefoxitin but it does not increase

the turnaround time (TAT) (Zurita, 2010), (Marlowe

and Bankowski, 2011). Therefore, for MRSA

identification, BSAC (British Society for

Antimicrobial Chemotherapy) recommends dilution

or disc diffusion of Columbia or Muller Hinton agar

with NaCl (2%) and incubation for 24 hours.

Nevertheless, because of its highly sensitive (>98%),

chromogenic agar is also commonly used for rapid

MRSA screening. Furthermore, FDA-approved

method for PCR and chromogenic agar only

available for nasal swabs, because of its different

result in each sample locations (Nathwani, 2008).

Selection of antibiotics for plating is also

essential since it determines the outcome of culture.

A study conducted by (Yamada, 2010) tried to

compare sensitivity and specificity among media

containing certain antibiotics, cefoxitin-based agar

media had 100 % sensitivities at 24 hours culture,

while lower results were found on the media

containing oxacillin or ceftizoxime. Consequently,

cefoxitin-based agar also is commonly used as first

option antimicrobial containing media for MRSA

screening while oxacillin as second option (Smyth

and Kahlmeter, 2005).

We conducted the study by using chromogenic

media and modified-conventional method, similar

results were found between the two-screening

method, by adding cefoxitin disc diffusion its

increased MRSA detection rate (p-value <0.001,

kappa index= 1.00) than conventional method alone.

(Han, 2007) evaluated mannitol salt agar (MSA) and

chromogenic media (CHROMagar Staph. aureus and

CHROMagar MRSA) for Staphylococcus aureus

detection, chromogenic media had shown higher

sensitivity at 24 hours (90.2 versus 76.5 % at 24

hours, p-value= 0.11), it is profound that the result

showed statistically non-significant, while using

MSA had higher specificity than chromogenic media

at 24 hours, 99.6% and 99.3 % respectively. But the

study only compared between two chromogenic-

based media for MRSA detection. In contrast, a

study conducted by (Patil and Gadagil, 2016) stated

that cefoxitin disc diffusion better than oxacillin and

chromogenic media, they carried out 200 clinical

isolates of Staphylococcus aureus and found 100 %

of sensitivity and specificity by using MSA-CFOX,

ICOSTEERR 2018 - International Conference of Science, Technology, Engineering, Environmental and Ramiﬁcation Researches

478

meanwhile with similar sensitivity, chromogenic

media had lower specificity, because of the findings,

chromogenic media should be used carefully in

detecting MRSA, especially alone.

Reduction in workload and reporting time by

using rapid identification like chromogenic media is

evident in study conducted by Lagace´-Wiens et al.

(2008) but using chromogenic media as screening

method is quite challenging since it can only be used

for a short period of time because of having short

expired date and the price is still high. Therefore,

using alternative method for screening MRSA is a

basis of this study especially beneficial for low-cost

laboratory. Poojay and Bhandarkar (2015) reported

lower price could be obtained by using conventional

method (MSA, blood agar plate, gram stain, catalase

test, coagulase test, and screening test using

cefoxitin) than using chromogenic media.

Nevertheless, using chromogenic media still saves

more than 48 hours instead of its high cost.

Although it was proved in 2009, we also

ascertain that the data was still reliable to be

compared to recent studies because of inconsistency

of the result showed in several studies which

compared the detection rate of MRSA using MSA-

CFOX and chromogenic media.

4 CONCLUSIONS

To conclude, modified-conventional method, we

used MSA-CFOX, becomes promising method for

MRSA screening instead of another expensive

method. Similar findings, sensitivity and specificity,

were evident in our study between using modified–

conventional method and chromogenic media. In

addition, this study also did not escape the limitation

since its MRSA positivity only found in four

samples, furthermore larger study is also needed to

give the evidence that modified-conventional

method is able to become one of choice for MRSA

screening. Notwithstanding, even our study was

conducted in 2009, we also proved using modified-

conventional method is become considerable method

to detect MRSA in general population.

ACKNOWLEDGEMENTS

This study was conducted to fulfill the requirement

in obtaining master’s degree. We thank to the staff

of Department of Microbiology, Universitas

Padjajaran, Sunarjati Sudigdoadi, MD, MS., PhD.

for their effort and supervision in this study. This

study was also used as preliminary study on using

Mannitol-salt agar supplemented cefoxitin powder

which is supported by Talent Research Universitas

Sumatera Utara, Basic Research Scheme of Year

2018 with the title is validity conventional method

of modification to conventional method and

ChromID MRSA as identification media

methycillin-resistant of Staphylococcus aureus

(MRSA),Contract Number 248/UN5.2.3.1/PPM/KP-

TALENTA USU/2018.

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