The Application of Chromogenic Culture Media for Rapid Detection of Food and Water Borne Pathogen

One of the latest techniques that used in recent decade to rapid detection of pathogenic agent in water and food is chromogenic media. These media are very specific and their component act as substrate for specific enzyme and depending on enzyme exhibit special color. This study is an introduction to chromogenic media as a powerful tool to rapid identification of bacterial and fungal agent in water and food sample. In this survey we review studies on using chromogenic media in rapid identification pathogen microorganisms in water and food in recent decade. Studies in different countries confirmed that chromogenic media are specific, rapid and sensitive and by using of them the most important water and food borne pathogenic microorganism such as E.coli, Staphylococcus aureus, L. monocytogenes, Salmonella and Candida can identified easily. in compare with other diagnosis methods chromogenic media have more advantage and can be an appropriate alternative for conventional and routine procedure. Chromogenic media eliminate the need of subculture and further biochemical test for identification pathogenic agent and at the shortest period of time pathogenic agent can be identified. Key word: Chromogenic culture media Rapid diagnosis Pathogenic microorganism food and water INTRODUCTION in recent years for this purpose we can hint to Isolation and identification of pathogenic (Table 1). Hill believes that for each one of these microorganism is one of the most important aspects of technique equipped lab and experienced technician food hygiene. Today identification and enumerations of are necessary and have expensive performs cost beside microbes in food have done in two main techniques. each of them have their advantage and disadvantage Most of These conventional methods are time however using technique like PCR and ELISA Gradually consuming and in spite of low cost often have difficulties find their position in microorganism identification for microbiologist. These depletion specially when rapid method [3]. As the matter of fact, today necessity of result report have importance from medical or economical using rapid diagnosis method of food pathogen are view considered more significant [2]. These technique more needed than ever. Lately using chromogenic based on inoculating bacteria in various media and media is one of the rapid diagnostic methods that producing visible colony in solid selective media. In some introduced as appropriate alternative for conventional bacteria like Salmonella it is necessary to take steps of method in developed countries and applying these pre-enrichment, enrichment and selective plating. sensitive, accurate and specific methods in diagnosis Fortunately discovering rapid detecting methods have process is a turning point in analytical microbiology solved most of theses problem. These methods have and considered as powerful tools [18]. Although high accuracy, rapid and with the help of them in chromogenic culture media first time introduced in shortest period of time the microorganism can be 1979 by Rambach but it officially offered and produced identified [1, 2]. About modern techniques that applied since 1991 [23]. genetically, physical and immunological method Am-Euras. J. Agric. & Environ. Sci., 4 (6): 693-698, 2008 694 Table1: Latest method of identification and isolation food pathogen microorganism Method Name Technique 1 Physical methods(diagnosis of microbe cell or their activities) Impedance measurement, Flowcytometr direct microscopic examination 2 Chemical and biochemical methods ATP assay (Bioluminescence) and Determination of microbial metabolism 3 Immunological methods(based on antibody) Immuno fluorescence, ELISA Latex agglutination 4 Genetical method Polymerase chain reaction, DNA Hybridation In recent decades many research has been done in coli variety O157H7 is significant food born pathogenic relation to rapid diagnosis pathogenic agent in food and agent and can cause diarrhea, hemorrhagic colitis and water by chromogenic media and many paper has been uremic hemolytic (HUS).this bacteria caused many published [4, 13, 5, 30, 25, 20]. epidemic by contaminated food[6] In this study, we review latest decade studies on Conventional culture media using for E.coli is using chromogenic culture media in rapid detection of Mccankey sorbitol agar that have a low exclusivity and pathogenic microorganism in food and water in different high false positive rang. Also in case of long time countries. incubation color changing occurred in colonies that can Identification Water and Food Borne Pathogen by media has been made that rises possibility of isolation of Chromogenic Media: By using chromogenic culture this variety of E.coli. Some of the most important of this media following pathogenic microorganism can be media cultures are rain bow agar (Biolerg America), BCM identify: O157H7 (BiosynthesisSwiss).Chromagar O157(Chromagar Coliforms and E. Coli: Coliforms and specially E.coli exclusivity made it possible to identification this pathogen considered as microbial contamination marker in food and water and they presence in drink water and food indicate that this material are contaminated with other enteric pathogens, so its isolation and enumeration have great important in determination of foods hygiene[28]. For detecting Coliforms acid and gas producing from lactose is the base of most diagnosis method, but occasionally some fecal Coliforms specially E.coli in some cases because of lack of ferment hydrogenase enzyme not produce the gas and this cause to new enzyme definition, among the rest can mentioned to existence of -Dgalactosidase in Coliforms and -D glucoronidase in 94 to 96 percent of E.coli.Other species of E.coli doesn't produce this enzyme[21]. For evaluating the enzyme activity chromogenic material have been used. For instance with chromogenic (5-bromo 4-chloro 3-indol -D-gluconoride) activity of D-glucoronidase can be evaluated. Also chromogenic substances (5-brmo 6-chlro 3-indol-D-galactosidase) have been used for demonstrating existence of -Dgalactosidase. Based on new definition variety commercial culture media for detecting Coliform especially E.coli by using chromogenic media has been invented that make it possible to identification them simultaneously in food and water. For instance can be mentioned to culture media like LMX broth media, ready cult media, chromo cult media (Merck Germany), Coli id media (Biomerio France) (Manafi et al. 2004, manafi andromans 1988). Escherichia not easily identified[1]. Recently new selective culture France).late medium because have high sensitivity and bacteria easily. Sensitivity of this method is as high as 98% and purple colonies are identifiable.Other species of Escherichia coli are colorless or blue in this media[2,32,14]. Salmonella: Because routine and conventional culture media apply for Salmonella diagnosis employing non specific marker sulfide hydrogen producing(SH2) have poor exclusivity that lead to numerous false positive result.(like Citrobacter and Proteus)[2]. In Salmonella id media(sm-id)(Biomerio France) Salmonella colonies identification by red color, Coliform in purple or blue and proteus species are colorless [14,12,11]. Biochemical characterizes used in the media are acid formation from gluconat incorporation with neutral red indicator and a chromogenic substrate material that make it possible to different Salmonella from other Entrobacteriacea. Selective element in media consists of bile salt, brilliant green Rambach agar (Merck Germany) that synthesis from propilen glycol, peptone, yeast extract, sodium dexoxy colat and neutral red. Salmonella chromogenic culture media like Rambach agar and Salmonella ID thanks to the high selectivity power, make the lab technician needless to performing test on irrelevant colonies and hereby save sufficient time to concentrating on real Salmonella colonies. This feature specially has importance on salmonelosis outbreak. Above culture media are selective and permit to easily identify most of the Salmonella species based on distinct red colonies. Am-Euras. J. Agric. & Environ. Sci., 4 (6): 693-698, 2008 695 Table 2: identifiable microorganism by chromogenic media according to genus, color sensitivity and differentional ability No. Genus Colony color Sensitivity (%) Differentional ability (%) 1. S.aureus Purple and red 95.5 99.4 2. S.epidermidis Blue 95.5 99.4 3. E.coli O157 Purple 98.0 99.0 4. E.coli spp Blue or colorless 98.0 99.0 5. Salmonella Red 100.0 100.0 6. V. cholerae Blue 99.0 98.5 7. V. valnificus Green 99.0 98.5 8. V. parahaemolyticus Purple 99.0 98.5 9. L. monocytogenes Blue with colorless halo 100.0 100.0 10. L. innocua Blue without halo 100.0 100.0 11. C. albicans Green 99.0 99.0 12. C. tropica Metallic blue 99.0 99.0 13. C. krusei Pinkish Velvet 99.0 99.0 The main defect of rambakh agar is that Salmonella Listeria Monocytogenes: is a dangerous food poisoning typhe, Salmonella paratyphi and some scare species have agent and exhibit high resistance to environmental factor not detected. Beside that, -D-galactosidase producing like low temperature, dryness and heat and isolated Salmonella species (e.g. Salmonella Arizona) exhibits from many food even dairy pasteurized product [2]. purple bluish colonies on both media. Efficiency of Conventional culture media like Oxford Staphilococous Aureus: Staphylococcus aureus considered uncertainly. Beside the lab technician in infections are one of the worlds most prevalence food conservative method should perform a lot of consuming infection and also are among the most significant culture test for confirming the presumptive colonies. pathogenic agent in clinical and hospital infection. The Listeria chromogenic media cultures (Chromoagar) source of bacteria are skin, mucous and persons hand and with the help of advance technology make it possible many epidemic have been reported [25, 27]. significant to direct isolation Listeria in one step. This test method conventional culture media applied for identification this sensitivity is 100% and in L. monocytogenes have bacteria are potato dextrose agar (PDA) and Manitol special blue color and surrounded with white color salt agar(MSA) which they act based on high salt halo due the phospholipids activities, so easily identified. concentration and manitol fermentation and have a lot L.innocuu is blue (without halo) and other Listeria species of false positive and negative results. Also another are colorless [2,4,6,13,19]. current culture media (blood agar) because necessity of time consuming and expensive test like coagulase that Vibrio Cholerae: Vibrio cholerae,Vibrio Parahaemolyticus should be perform at least on 5 to 10 presumptive colonies and Vibrio vulnificus are pathogens that cause food from each sample is not routines [7,13]. While S.aureus borne disease, generally contagion by polluted water and chromogenic medias are unique with high sensitivity vegetable and lead to severe food poisoning [6,13,15]. more than 95% (equal to coagulase), make it possible to Conventionally TCBS culture media have been used for accurate and easy identification of the pathogens. isolation and identification which have poor sensitivity S.aureus`s colonies have 99.4% differentional ability and beside long turn around time, while specific Vibrio demonstrated with purple color and other staphylococcus chromogenic media (Chromoagarr vibrio) easily isolated species colonies are blue or colorless [6,7,12,14,22,31,30]. this 3 vibrio species from other species and the sensitivity In recent years more hospitals are involving with of the method evaluated 98.8%. In this media the color methicillin resistance S.aureus (MRSA)and current produced by Vibrio cholera, Vibrio parahaemolyticus culture media demonstrated unreliable result in detection and V. vulnificus colonies are blue, green and purple of these species while chromogenic media produced for respectively and other species are colorless [14]. resistance strain with high differential and sensitivity as 100% just in one 24 hours incubation can isolate the Yeast (Variety Species of Candida): Among the various pathogen and antibiotic sensitive strain not able to yeast Candida have significant rule in human infection growth in this media[21, 22, 24]..Diedern and others in and food spoilage and many report have been received 2006 applied new chromogenic media to detecting MSRA relate to Candida albicans infections[13]..yeast isolating and achieve the satisfactory results [15,17]. and diagnosis take a several day in lab and have not high and Palcam for culturing and isolating the organism Am-Euras. J. Agric. & Environ. Sci., 4 (6): 693-698, 2008 696 sensitivity, but chromagar Candida beside the ability to exclusivity and performance in compare with other detecting yeasts, have ability to simultaneously isolating technique. Rahbar and other (2008) for MRSA variety species of Candida according to their colonies identification have used four method and culture media color. Also this media can detect antifungal resistance that was consist Manitol salt agar, E-test Mic, Oxacillin species. Isolation ability in this method is 99% and screen agar, Plus and chromogenic culture media the Candida albicans species identified by green color, result showed chromogenic media in compare with other C. tropica metallic blue and C.krusei pink [15] in Table 2, method have had higher sensitivity and exclusivity as identifiable microorganism by chromogenic culture media sensitivity and exclusivity of this method defined 100% had been demonstrated. [2, 14, 22, 25]. The result of this study are similar to other RESULTS AND DISCUSSION It is clear that these techniques like other methods Conventional methods for detecting pathogen agent dissimilar usages are the most important of this method generally based on bacteria culture in different media disadvantages. For instance ready cult media can be used and visible colonies production in a selective solid media only for drink water and can not be used for food and conducting biochemical test in liquid media. These specimen. In this method identification of some genus methods are time consuming and for bacteria isolation of bacteria is not feasible for example clostridium in food sometimes need to perform following step of perfringens, because often water contaminated with pre-enrichment, enrichment and culture in selective solid variety species of this bacteria after culture in media and occasionally it is necessary to employed chromogenic media there will be a lot of red colors so its further test to confirm like biochemistry test e.g. catalase, not possible to identification bacteria species because oxidize, coagulase and IMVIC test[2]. in recent years all of the red colonies are not clostridium perfringens. biotechnology advance lead to change in food test Beside ammonium hydroxide presence in the media is technique and today, we benefit from methods that are reaky and sometimes the ammoniums itself eliminate the more specific, faster and often more sensitive in compare colonies. this problems lead to new chromogenic culture with conventional method [22, 23]. One of the latest media production that are able differ clostridium method introduced for rapid detection of pathogen species (C.P. chromagar). Late media have ability to microorganism in food is using chromogenic media. produce variety of color and further research have Utilizing these media can eliminate necessity of further been conducted related to this media [18]. subculture and biochemical test in identification process One of the important aspect these media come of bacteria. these technique based on production useful is water contamination rapid identification and substrate material for specific microorganism enzyme, utilize this methods have huge impact on prevention of according to the produced color the microorganism can water borne diseases.2.1 million people have died hence be identified easily [13]. using contaminate water WHO announced. These issues As the matter of fact many substance produce especially in crises and military have great importance, colored or fluorescent substance after reaction with because in such situation water microbial test result microbe enzyme or other component this feature has should reported at possible shortest period of time been used for identification. Most of the substrate utilize chromogenic media are very helpful. In conventional for chromogenic media are phenol and indol derivatives E.coli and Coliforms (that are water contamination and often Comarin derivatives have been used for indicator) identification methods are very time consuming fluorescent substrate. Derivatives of -D-gluconoride furthermore there are many bacteria genus that are or -D-galactoronid widely use in food pathogen able to produce acid from lactose (ie. Klebsiella, rapid diagnostic kit. These component metabolized by Citrobacter, Entrobacter and etc) that their presence in respective decomposer microbial enzyme chromogenic or water not mandatory result of contamination [4,5]. So we florescent product, produced from their metabolism[14]. need rapid and specific method that be able easily Chromogenic media have many advantages like rapid identified Coliforms specially E.coli. detection, high sensitivity, highly specific, needles to Our research showed according to these bacteria further biochemical test in microorganism identification. importance as indicator of food and bacteria Research conducted all over the world has been contamination many research has been done on them. shown that these technique have higher specifically, Herein Anfor and others tested 2500 water sample. In all survey [12, 15, 21, 22, 26, 30]. have defect. Specific media culture, expensive cost, Am-Euras. J. Agric. & Environ. Sci., 4 (6): 693-698, 2008 697of cases test results was satisfactory and even variety interpretation [14, 15].The result of this reviewed studiesspecies of E.coli have been identified [20,22] for according to the last decade research all over the worldthis purpose LMX and ready cult culture media [14, 15, 18, 22, 23, 30] chromogenic culture media areemployed that make it possible to rapid and useful tool in rapid identification of food and watersimultaneous detection of E.coli and Coliforms in unite pathogen, and with utilizing them significant food andculture media by consisting of following compound water pathogenic microorganism i.e. Salmonella, E.coli,(5 bromo 4 chloro 3 indol -D-galactopyranose) and S.aureus monocytogenes and Candida can be easilyfluorogenic component 4 methyl -D-gluconoride. detected. In comparison with other method chromogenicAlso adding 1-isopropil -D-thiogalactopyranose (IPTG) media are more rapid, accurate and reliable and cansubstance that easily produces lactose yield to produce utilize as alternative for conventional methods. Henceand increase activity of galactosidase. in this method employing these media eliminate the need of subculture99% of Escherichia coli can be detected and identified and further biochemical test for identification ofexclusively.pathogenic agent and at the shortest period of timeIn research conducted by Manafi and others on possible, pathogenic agent can be identified. This feature1246 sample contaminated to varies species, 1240 species especially in in disasters and military condition like(99%) have been identified by chromogenic media and a maneuver and military camps have special important forfew non Coliform bacteria such as Serratia, Hafnia, Vibrio preventing food and water borne outbreakand Aeromonas species have and false positive result. hebelieves that employing chromogenic media in compareREFERENCESwith the other standard methods detects more Coliformsand E.coli within 24 hours[8]. Baunadonna and others 1. Vonderzant, C. and D.F. Splittstoesser, 2004.studies (2007) showen this media when compared withCompendium of methods for microbiologicalother method and confirmatory tests, chromogenic mediaExamination of foods (APHA), 36: 605-609.have more sensitivity when identifying E.coli in water 2. Tavakoli, Hamidreza, 2006. PCR and its applicationsample[14, 6]. Pitkanen and others confirmed ability ofin rapid detection pathogen agent in food. 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