Journal of Food: Microbiology, Safety & Hygiene
Open Access

An Evaluation on the Recovery of Staphylococcus aureus in CHROMagar Versus Conventional Selective Agar in Food Sample Analysis

Lei Benitez^{* *}Correspondence: Lei Benitez, Department of Food Microbiology, University of Hamburg, Hamburg, Germany, Email:

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Introduction

The development and implementation of novel diagnostic techniques has had a profound effect on microbiology laboratory services in recent decades. Current follow-up culture-based isolation of the food microbes can be labor-intensive and time- consuming due to the long incubation period. In some cases, such isolation is even unsuccessful due to lack of sufficiently selective isolation media and tedious biochemical tests. Recently, media allowing the detection of specific bacteria genus by the incorporation of chromogenic substrates have been introduced. Compared to conventional selective media chromogenic media had higher specificities, with more false-negative results.

Therefore, any new medium or method with the ability to streamline culture processing in a meaningful way, such as reducing technologist workload, improving result Turn Around Times (TATs), or reducing laboratory costs, would be welcomed and has the potential to have considerable laboratory impact. The conventional methods for detecting the foodborne bacterial pathogens present in food are based on culturing the microorganisms on agar plates followed by standard biochemical identifications [1]. Conventional methods are usually inexpensive and simple but these methods can be time consuming as they depend on the ability of the microorganisms to grow in different culture media such as pre-enrichment media, selective enrichment media and selective plating media. Problems associated with the direct culture of microbial species include difficulties in identification of pathogens, the time and expense involved in identifying and typing of pathogens isolates and the effect of selective media and/or selective isolation methods [2].

In recent years, the use of chromogenic media has become a key method for the rapid identification of microorganisms. These media detect key microbial enzymes as diagnostic markers for pathogens through the use of “chromogenic” substrates incorporated into a solid-agar-based matrix [3]. Recently in the United States of America and United Kingdom, and more recently in Australia, chromogenic substrate technologies have been accepted as standard methods [4-7]. Over the last two decades, there has been a rapid expansion in the development and commercial availability of chromogenic agar media for the detection of pathogenic bacteria and yeasts [3,8].

In contrast to conventional culture media, chromogenic media allow direct colony color-based identification of the pathogen from the primary culture. This reduces the need for subculture for further biochemical testing and hence the time until a result is obtained [9]. Chromogenic media are intended to correctly identify more-frequently occurring bacteria and yeasts or organism groups on primary culture with no further testing or a minimum number of confirmatory tests [10]. Target enzymes hydrolyze chromogenic substrates generating colored products which allow for easy identification of specific organisms [3,11]. This facilitates the differentiation of species within polymicrobial cultures and the targeting of pathogens with high specificity. When specific pathogens are targeted, selective agents such as antibiotics are employed to limit the number of species able to grow. Most media rely upon the inclusion of indoxylic substrates in order to generate colonies with contrasting colors. For example, the release of green and red chromogens from two distinct substrates can result in the formation of green, red or purple colonies depending on whether one or both enzyme activities are present [12]. Pathogens may therefore be differentiated from commensal bacteria by their possession of either one or both enzymes [12,13].

The purpose of this study is to evaluate the accuracy, specificity, sensitivity and selectivity of the CHROMagar media for Staphylococcus aureus when compared with conventional selective agar as specified in international standards in different types of food. In addition, the limit of detection for the chromogenic media was investigated in this study.

Materials and Methods

Strains

A total 10 bacterial strains included in the study which includes lab isolates from natural samples, isolates from proficiency samples and quality control strains from National Collection of Type Cultures (NCTC) and American Type Culture Collection (ATCC). All strains were identified by VITEK 2 Compact system or respective biochemical tests (Table 1).

Table 1: Parameters ofinclusivity strains and exclusivity strains.

Culture media

Chromogenic media was purchased as powdered media from CHROMagar^TM through Biomed Dubai. The plates were prepared according to the manufacturers' instructions. Briefly, weight as specified by grams of dehydrated media were reconstituted in 1 liter of distilled water. They were brought to boil by repeated heating's and then cooled in a water bath at 45°Cbyswirlingandstirring.Then 20ml of the media were dispensed into petri dishes. They were kept in the dark at 4°C and used for further studies.

Baird Parker Agar (Biolife Italiana Srl º491116) for Staphylococcus aureus was used as conventional agar as per the standard ISO method. All medias were prepared as per manufacturer's instructions and stored at 4°C and used for further studies.

Identification system

All isolates were identified using VITEK 2 compact system using respective cards such as GN for Salmonella, Cronobacter sakazakii. GP cards used for Staphylococcus aureus and Listeria monocytogenes identification. Bacillus Identification Card (BCL) VITEK cards used to identify Bacillus species.

Matrixes

Different matrixes were used in the study based on the strains of choice and the regulatory requirements. Details as mentioned on below Table 2.

Table 2: Microbiological criteria for food stuffs and details of the matrixes selected for each parameter.

Inoculum preparation

Purified isolates of all strains were prepared by sub culturing the reference strains and lab isolates onto plate count agar and incubation at 37°C for 24 hrs. A standard turbid preparation of 0.5 for each microbes prepared with McFarland standard using Densi Check plus (Biomeriux). Further serial dilutions conducted to achieve the desired concentration of inoculum. Three different inoculum levels prepared for each strains (Low (5-10 cfu), Medium (50-100) and High (1000 and above)). After appropriate dilutions, each desired dilution of each of the isolates was plated onto the five chromogenic media and onto standard agar, which served as a control.

Food sample preparation

A wide variety of food samples selected based on the UAE.S GSO 1016/2017, microbiological criteria for food stuffs. Details of the matrixes selected as shown in Table 2.

Test method

For Staphylococcus aureus, quantitative techniques embraced to identify the sensitivity, specificity and accuracy of test methods. Sample preparation was performed based on ISO-6887-2 (Table 3).

Table 3: Comparison between chrom agar and Standard method media.

For quantitative analysis, 20 gm of sample weighed in a sterile stomacher bag for each food matrix aseptically in quadruplicates. An initial 1:10 dilution was prepared for sample by adding 180 ml of maximum recovery diluent (Biolife EG7204) in each. Among the four parts weighed 3 were spiked with low, high and medium level of inoculum of each organism under study and one sample kept as control. Homogenize the sample using a stomacher (Smasher, AES #44497426) for 30 seconds, Later on 0.1 ml of Inoculum were added to the CHROMagar plates and corresponding standard control plates as mentioned in (Table 2), and incubated for 24 hrs ± 2 h at 37 ± 1°C for Staphylococcus aureus. 25 gm of each sample weighed in a sterile stomacher bag for each food matrix aseptically in quadruplicates. An initial 1:10 dilution was prepared for sample by adding 225 ml of maximum recovery diluent in each. Among the four parts weighed 3 were spiked with low, high and for quantitative analysis, colonies with typical morphology for each plates were counted and recorded separately. 5 presumptive colonies of each plates were purified in plate count agar for further confirmation. Confirmation was done by conventional techniques based on International Standard Organization (ISO) standards and on VITEK 2 Compact-Biomeriux-VK2C5407 according to the manufacturer’s instructions (Table 4).

Table 4: Confirmation techniques and kits used for identifying Staphylococcus aureus.

Statistical analysis

Results were analysed statistically to identify accuracy, specificity, sensitivity and selectivity of alternative methods. Precision was calculated trough the comparison of test results with the results of reference method as well as with proficiency sample. Assessment of the sensitivity, specificity and selectivity of CHROMagar was achieved through the identification of typical colonies isolates from CHROMagar.

Results and Disussion

In total 97 samples were analyzed, On CHROMagar S. aureus, all typical colonies appeared as mauve colonies after 24 h of incubation compared with grey to black colonies with and without zone on Baird Parker agar after incubation for 48 h. Most of the competitive flora are inhibited and some appeared as blue color colonies in the CHROMagar. False positive result was observed with Staphylococcus warneri which appeared as mauve coloured and indistinguishable from the Staphylococcus aureus on CHROMagar. But the strain was identified later as coagulase negative in Latex Agglutination Kit for S. aureus (MASTSTAPH).

Precision

Precision (accuracy/trueness) of the test method using CHROMagar was determined by estimating the mean percentage recovery of test results in comparison with Baird Parker agar. A mean recovery of 95 % shows the high accuracy of test results, and therefore there is no indication of significant bias. At all levels of inoculums from low to high the recovery was more or less similar (Table 5).

Table 5:  Comparison of bacterial recovery rates from various food samples using BPA and CHROMagar staph aureus media.

Accuracy, sensitivity, specificity and selectivity

The outcomes of the analyses of the data show that for S. aureus, the CHROMagar^TM method is sensitive and specific. 6 strains of S. aureus tested were detected by the alternative method. Of the 5 non-target strains tested, cross-reactivity observed only with Staphylococcus warneri in the alternative method. All S. aureus had a characteristic profile on CHROMagar^TM. The confirmation was done by coagulase test Mast Latex Agglutination Kit (MLAK). Colonies showing typical morphology and negative coagulase test were subjected for VITEK 2 confirmation and identified as Staphylococcus warneri (Table 6-9).

Table 6: Confirmation of mauve colored colonies in various strains of staphylococcus aureus using LATEX

Table 7: Exclusivity testing of various bacterial strains on selective media, comparing expected colony morphology with actual results and confirmation by LATEX agglutination test.

Table 8: Results of inclusivity and exclusivity tests.

Table 9: Accuracy, sensitivity, specificity and selectivity.

Conclusion

Out of 97 total samples tested for exclusivity and inclusivity studies, 55 target colonies shown typical characteristics and confirmed as Staphylococcus aureus by mast staph latex agglutination kit. Most of the non-target strains suppressed and some shown atypical colonies on CHROMagar. However, the possibilities of coagulase negative Staphylococcus species shows typical colonies in CHROMagar still exists and can be rectified by adding the confirmation step of coagulase test. We conclude that CHROMagar is highly effective for the isolation and presumptive identification of S. aureus from food samples and that it compares favorably with conventional media. The preparation and use of CHROMagar S. aureus is much less labor intensive than conventional methods and requires fewer reagents for confirmation of suspect colonies of S. aureus as the cases of false positive and negative were less. CHROMagar for Staphylococcus aureus was also highly specific (97.94%), with few other species resembling S. aureus. The specificity increased from 95.24% to 100% when the result was evaluated in conjunction with mast latex kit. In the present study, one of the biggest advantage observed was the shortest Incubation period of 24 hrs with CHROMagar yields a highest recovery rate of 95% over the conventional media which required 48 hrs of Incubation as per the standard method for food analysis. The selectivity was -2 which was considerably better than the guidance value of -1 suggested by ISO/TR 13843 for colony count methods.

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