Chromogenic Medium Research Articles

Evaluation of Selective-Enrichment and Chromogenic Media for Salmonella Detection in Raw Shell Egg Contents with a Low Microbial Load.

The current study was conducted to evaluate the ability to recover Salmonella from shell egg contents by culture methods. A total of 4,000 eggs were obtained from a grading and packing center located in the Gyeonggi Province of South Korea, and 200 samples were created by pooling 20 broken eggs. The pooled samples were held at room temperature for 4 d before a 25-mL aliquot of each pool was added to 225 mL of modified trypticase soy broth (mTSB) and incubated at 35°C for 24 ± 2 h. A loopful of the culture was streaked onto chromogenic Druggan-Forsythe-Iversen (DFI) agar and incubated at 36 ± 1°C for 18-24 h. In addition, 1 mL and/or 0.1 mL of the mTSB cultures were added to 10 mL of Muller-Kauffmann tetrathionate with novobiocin (MKTTn) or Rappaport-Vassiliadis (RV) broth, and they were incubated for 24 ± 2 h at 35 ± 2°C or 42 ± 0.2°C, respectively. A loopful from these cultures was streaked onto Brilliant Green (BG), xylose lysine deoxycholate (XLD), and bismuth sulfite (BS) agar plates, respectively. Directly streaking onto DFI agar revealed the presence of Salmonella in 14 out of the 200 pooled samples (7%); whereas the combination of RV medium and BG, XLD, and BS agar detected the pathogen in only 9 (4.5%), 7 (3.5%), and 3 (1.5%) of the pooled samples, respectively. When MKTTn broth was used, Salmonella was detected in 7 (3.5%), 2 (1%), and 0 (0%) of the samples when streaked onto BG, XLD, and BS agar, respectively. The results indicate that direct plating onto DFI agar without enrichment was the most suitable among the methods evaluated in this study for detecting Salmonella in raw shell egg contents with a low microbial load.

Household Microbial Water Quality Testing in a Peruvian Demographic and Health Survey: Evaluation of the Compartment Bag Test for Escherichia coli.

AbstractThe Joint Monitoring Program relies on household surveys to classify access to improved water sources instead of measuring microbiological quality. The aim of this research was to pilot a novel test for Escherichia coli quantification of household drinking water in the 2011 Demographic and Health Survey (DHS) in Peru. In the Compartment Bag Test (CBT), a 100-mL water sample is supplemented with chromogenic medium to support the growth of E. coli, poured into a bag with compartments, and incubated. A color change indicates E. coli growth, and the concentration of E. coli/100 mL is estimated as a most probable number. Triplicate water samples from 704 households were collected; one sample was analyzed in the field using the CBT, another replicate sample using the CBT was analyzed by reference laboratories, and one sample using membrane filtration (MF) was analyzed by reference laboratories. There were no statistically significant differences in E. coli concentrations between the field and laboratory CBT results, or when compared with MF results. These results suggest that the CBT for E. coli is an effective method to quantify fecal bacteria in household drinking water. The CBT can be incorporated into DHS and other national household surveys as a direct measure of drinking water safety based on microbial quality to better document access to safe drinking water.

Fast methods of fungal and bacterial identification. MALDI-TOF mass spectrometry, chromogenic media

MALDI-TOF mass spectrometry is now a routine resource in Clinical Microbiology, because of its speed and reliability in the identification of microorganisms. Its performance in the identification of bacteria and yeasts is perfectly contrasted. The identification of mycobacteria and moulds is more complex, due to the heterogeneity of spectra within each species. The methodology is somewhat more complex, and expanding the size of species libraries, and the number of spectra of each species, will be crucial to achieve greater efficiency. Direct identification from blood cultures has been implemented, since its contribution to the management of severe patients is evident, but its application to other samples is more complex.Chromogenic media have also contributed to the rapid diagnosis in both bacteria and yeast, since they accelerate the diagnosis, facilitate the detection of mixed cultures and allow rapid diagnosis of resistant species.

Evaluation of Chromogenic VRE Medium versus Conventional Vancomycin E Test in Detection of Vancomycin Resistant Enterococci

Background : Enterococci have become resistant to a wide range of antibiotics which include glycopeptides like vancomycin. The rapid increase of vancomycin resistance enterococci (VRE) compromises physicians to treat infections caused by these strains as the therapeutic options for VRE infections are very limited. Objectives : To evaluate the efficacy of chromogenic VRE medium in detection and identification of vancomycin resistant enterococci. Methodology : the present study was conducted in Al Abbassia Fever Hospital, El Sayed Galal Hospital, Tanta University Hospital and Kafr El Sheikh General Hospital. Sixty enterococcal isolates were collected (thirty vancomycin susceptible, eight vancomycin intermediate resistant and twenty two vancomycin resistant isolates). Antibiotic susceptibility pattern for enterococcal isolates was done using disc diffusion method, chromogenic medium and MIC of vancomycin for vancomycin resistant and intermediate resistant isolates was determined by E test. Results : The most common isolated vancomycin resistant and intermediate resistant species was E. faecium (53.1%) followed by E. faecalis (40.6%). The highest rate of isolation of VRE was from urine (50.0%). VRE isolates were mostly recovered from ICUs (45.5), 41.7%, of the collected isolates were vancomycin resistant by disc diffusion method. Regarding E-test, out of fourty two vancomycin resistant and intermediate resistant by disc diffusion method 52.4% were resistant and 19% were intermediate resistant. HiChrome VRE had 100.0% sensitivity and 83.3% specificity in identifying vancomycin resistant enterococcal isolates. Conclusion: We can depend on chromogenic media in both detection and identification of vancomycin resistant enterococci.

Detection of Extended Spectrum Beta Lactamase Producing Strains among Clinical Isolates of Escherichia Coli and Klebsiella Pneumoniae in Alexandria Using Chrom-ID ESBL Agar and Molecular Techniques

Background: Antibiotic resistance has become a serious global problem and affects almost every bacterial species for which treatment with antibiotics is available. The increased prevalence of Enterobacteriaceae producing ESBLs creates a great need for identifying these organisms for infection control and epidemiological surveillance. Objectives: In this study, the aim was to (i) evaluate different phenotypic methods for detection of ESBL producing E. coli and K. pneumoniae (ii) Evaluate the diagnostic performance of the selective chromogenic culture media, chromID ESBL, (iii) Determine the prevalence of the genes blaTEM, blaSHV, and blaCTX-M, which are responsible for ESBL production in clinical isolates of E. coli and K. pneumoniae. (iv)Identify the sequence types of the most prevalent ESBL genes among E. coli and K. pneumoniae in Alexandria. Methodology: One hundred isolates of ESBL producing E. coli and K. pneumoniae isolates were collected. Phenotypic characterization of ESBLs by combined disc test, double disc synergy test (DDST), and the modified double disc synergy test (MDDST) was performed simultaneously with antibiotic susceptibility testing. ChromID ESBL agar (bioMerieux) was evaluated for ESBL detection. ESBLs positive strains were tested for the presence of ESBL encoding genes by PCR. After amplification, twelve TEM, SHV and CTX-M genes were subjected to nucleic acid sequencing. Results: An ESBL phenotype was confirmed in 100/173 (57.8%) isolates by combined disc test. The sensitivity of DDST was 44% when the distance kept at 20 mm. Cefepime yielded the highest performance among the β-lactams, with sensitivity 70% when tested with amoxicillin-clavulanate and 86% when tested with piperacillin-tazobactam. The highest sensitivity among phenotypic methods (98%) was obtained with chromID ESBL. PCR of TEM, SHV and CTX-M genes revealed that 80% were positive for at least one of the studied genes. Sequencing of the randomly selected genes revealed that the eight bla TEM amplification products were TEM-1, while the two bla SHV were SHV-11 and the two bla CTX-M genes were CTX-M-15. Conclusion: using only one disk combination might fail to detect ESBL production. The MDDST using cefepime and piperacillin-tazobactam gives the highest sensitivity followed by MDDST using cefepime and amoxicillin-clavulanate. Agars with chromogenic substrates could be applicable as screening methods for ESBLs producing strains.

Identification of microorganisms grown on chromogenic media by MALDI-TOF MS

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and chromogenic media are widely used in clinical microbiology laboratories to facilitate the rapid selection and identification of pathogens. The aim of this study was to evaluate whether usage of chromogenic media limits the diagnostic performance of MALDI-TOF MS for microbial identification. A total of 386 microorganisms collected and analyzed at five laboratories were included. Isolates were cultured on relevant chromogenic media and non-selective agar plates in parallel and identified using the Bruker MALDI-TOF MS. Among the tested isolates, no misidentification was recorded and there was no medium-related difference in the identification level. However, score values were overall slightly but significantly lower for isolates grown on chromogenic media. In conclusion, the use of chromogenic culture media tested here had no relevant impact on MALDI-TOF MS performance for diagnostic purposes.

Efficacy and acceptability of rectal and perineal sampling for identifying gastrointestinal colonization with extended spectrum β-lactamase Enterobacteriaceae

ObjectivesWe evaluated ‘pre-laboratory’ factors associated with the detection of extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) colonization including anatomical site, and staff and patient factors. MethodsAll admissions to a large London hospital over 3 months were approached to provide rectal and perineal swabs, which were cultured for ESBL-E using chromogenic media. ESBL-E detection rates for patient- or staff-collected rectal or perineal swabs were compared using McNemar tests. Binary logistic regression was used to explore factors associated with patients declining to provide a rectal swab. The impact of simplifying the verbal study description to patients to improve the participation rate was evaluated. ResultsCarriage of ESBL-E was significantly higher in rectal swabs than perineal swabs (7.8% of 4006 versus 3.8% of 4006, p <0.001), whether collected by staff or patients; 31.9% of 869 patients did not provide a rectal swab before the change in study description compared with 7.6% of 3690 patients afterwards (p <0.001). In multivariable analysis, factors associated with patients declining to provide a rectal swab were younger age (OR 0.99, 95% CI 0.99–1.00), female gender (OR 1.26, 95% CI 1.04–1.52), transfers from other hospitals (OR 1.77, 95% CI 1.07–2.93) or an unknown admission route (OR 1.61, 95% CI 1.09–2.37), being admitted before the change in study description (OR 0.39, 95% CI 0.31–0.48), and the staff member who consented the patient (p <0.001); ethnicity was not a significant factor. ConclusionsRectal swabs are recommended for the detection of ESBL-E colonization. Staff and patient factors influence whether patients participate in prevalence studies, which may skew their findings.

Evaluation of Chromogenic agar media for rapid identification of Candida species.

Rapid identification of Candida isolates to the species level is essential in order to optimize the antifungal treatment. This study aimed to isolate and identify different species of Candida from various clinical specimens and to evaluate the use of chromogenic agar media as primary culture media for culture of Candida as well as for rapid identification of Candida species. A total of 100 different clinical specimens were studied (oral swab 35, high vaginal swab 28, catheterized urine 15, nail 14, bronchoalveolar lavage 04 and peritoneal fluid 04). Isolation of Candida species was done by primary culture in Sabouraud Dextrose Agar (SDA). Subsequent identification of species was performed by germ tube test, carbohydrate assimilation test (with commonly used twelve sugars) and subculture in chromogenic agar medium. Out of 64 isolated Candida, C. albicans 33 (51.53%) was the most predominant Candida species followed by C. tropicalis 17 (26.56%). The species of C. glabrata was 4 (6.25%), C. parapsilosis 4 (6.25%), C. krusei 3 (4.68%) and C.guilliermondii 2 (3.2%). One of the isolated Candida species was unidentified. The sensitivity and specificity of chromogenic agar media for C. albicans were as 96.97% and 96.87% respectively. The sensitivity and specificity for C. tropicalis were 94.12% and 97.87% respectively. C. krusei and C. glabrata both showed 100% sensitivity and specificity on chromogenic agar media. Efficacy of chromogenic agar media is nearly similar to carbohydrate assimilation method in species identification of Candida.Bangladesh J Med Microbiol 2015; 9 (1): 22-26

A Decade of Development of Chromogenic Culture Media for Clinical Microbiology in an Era of Molecular Diagnostics.

In the last 25 years, chromogenic culture media have found widespread application in diagnostic clinical microbiology. In the last decade, the range of media available to clinical laboratories has expanded greatly, allowing specific detection of additional pathogens, including Pseudomonas aeruginosa, group B streptococci, Clostridium difficile, Campylobacter spp., and Yersinia enterocolitica. New media have also been developed to screen for pathogens with acquired antimicrobial resistance, including vancomycin-resistant enterococci, carbapenem-resistant Acinetobacter spp., and Enterobacteriaceae with extended-spectrum β-lactamases and carbapenemases. This review seeks to explore the utility of chromogenic media in clinical microbiology, with particular attention given to media that have been commercialized in the last decade. The impact of laboratory automation and complementary technologies such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is also assessed. Finally, the review also seeks to demarcate the role of chromogenic media in an era of molecular diagnostics.

C. difficile PCR-ribotype 023 might go undetected when using ChromId C. difficile agar

We compared the performance of the new chromogenic medium ChromID C. difficile with that of CLO agar. ChromID C. difficile agar is a sensitive medium that can accelerate the presumptive identification of C. difficile, however ribotype 023 might go undetected when using this chromogenic medium.

Phenotypic and genotypic detection of Candida albicans and Candida dubliniensis strains isolated from oral mucosa of AIDS pediatric patients.

ABSTRACTThe aim of this study was to assess a collection of yeasts to verify the presence of Candida dubliniensis among strains isolated from the oral mucosa of AIDS pediatric patients which were initially characterized as Candida albicans by the traditional phenotypic method, as well as to evaluate the main phenotypic methods used in the discrimination between the two species and confirm the identification through genotypic techniques, i.e., DNA sequencing. Twenty-nine samples of C. albicans isolated from this population and kept in a fungi collection were evaluated and re-characterized. In order to differentiate the two species, phenotypic tests (Thermotolerance tests, Chromogenic medium, Staib agar, Tobacco agar, Hypertonic medium) were performed and genotypic techniques using DNA sequencing were employed for confirmation of isolated species. Susceptibility and specificity were calculated for each test. No phenotypic test alone was sufficient to provide definitive identification of C. dubliniensis or C. albicans, as opposed to results of molecular tests. After amplification and sequencing of specific regions of the 29 studied strains, 93.1% of the isolates were identified as C. albicans and 6.9% as C. dubliniensis. The Staib agar assay showed a higher susceptibility (96.3%) in comparison with other phenotypic techniques. Therefore, genotypic methods are indispensable for the conclusive identification and differentiation between these species.

Antifungal Drug Resistance among Candida albicans and Non-albicans Candida Species Isolates from a Tertiary Care Centre at Allahabad

Objective: The aim of our study was to identify the distribution of Candida species among clinical isolates and their sensitivity pattern for common antifungal drugs. Methods: This descriptive study was carried out in a tertiary care hospital in Allahabad. A total of One hundred and four Candida isolates were included in the study. Identification and speciation of the isolates were carried out by Germ-tube test, Chlamydoconidia production test, colony characterization in chromogenic agar medium, carbohydrate assimilation test and growth at 45°C. Four antifungal drugs such as Fluconazole (25 mcg), Clotrimazole (10 mcg), Nystatin (100 U) and Amphotericin-B (100 U) were tested by disk diffusion method. Descriptive statistics was used which involves the use of simple percentage and bar chart to analyze the data. In addition, Chi-square test was performed and P value was calculated. Results: Out of 104 Candida isolates, 60 (58%) were from Urine, 36 (34.5%) were from Sputum and 8(7.5%) were from blood isolates. Of the total of 104 isolates 64 (61.5%) were Candida albicans, 20(19%) Candida dubliniensis, 10 (9%) Candida tropicalis, 7(6.5%) Candida parapsilosis and 4(4%) Candida glabrata respectively. Resistance rates for commonly use antifungal drugs among the isolates are as followed Fluconazole 76 (73%), Amphotericin B 40 (38.5%), Nystatin 4(4%) and Clotrimazole 60 (58%) respectively. For Candida albicans and Candida dubliniensis most active antifungal drug was Nystatin (93%; 100% respectively). Discussion: Species level identification of Candida and their antifungal sensitivity testing should be performed routinely in all microbiology laboratories to prevent the spread of antifungal resistance.

Fungal Profile of Vulvovaginal Candidiasis in a Tertiary Care Hospital.

Vulvovaginal Candidiasis (VVC) is a common medical health problem of adult women. It is most commonly caused by Candida albicans. But there is a change in fungal profile. Sabouraud's Dextrose Agar (SDA) is the most common culture medium used where mixed fungal infection may be missed. It can be detected easily by using chromogenic culture medium. To know the fungal profile of vulvovaginal candidiasis using Candida CHROMagar and antifungal susceptibility pattern in patients attending tertiary care hospital. Culture confirmed cases of VVC presented at Department of Obstetrics and Gynaecology of Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India, from July 2015 to December 2015 were included in the cross-sectional study. Two high vaginal swabs were collected and inoculated on SDA and Candida CHROMagar (Hi-Media, Mumbai, India). After overnight incubation the colonies were counted and colour of the colonies were recorded from Candida CHROMagar. Candida spp. were identified by sugar fermentation and assimilation tests and other conventional tests. Antifungal susceptibility tests were performed by the disc diffusion method using fluconazole (25 μg) and voriconazole (1μg) as per the Clinical and Laboratory Standards Institute (CLSI - M44-A2) guidelines. A total of 50 culture confirmed (23.7%) cases were detected from 211 clinically suspected VVC cases. Candida glabrata (45.1%) was the most common isolate, followed by Candidatropicalis (23.5%), Candida albicans (17.6%), Candida krusei (9.8%) and Candida parapsilosis (3.9%). One mixed infection of C. glabrata and C. albicans was identified on Candida CHROMagar. Mixed fungal infection was observed in 2% of positive culture and 0.5% of VVC cases. The antifungal susceptibility testing revealed that 15.7% and 9.8% isolates of Candida spp. were resistant and Susceptible Dose Dependent (S-DD) respectively to fluconazole. The increase resistant against fluconazole was because of increased isolation of C. glabrata strains. All strains of Candida spp. were susceptible to voriconazole. C. glabrata was the most common causative agent of VVC in a tertiary care hospital. Chromogenic culture medium facilitates detection of mixed fungal infection. In vitro susceptibility testing should be used to guide the treatment especially in cases of non-albicans Candidiasis.

Faecal Carriage of Gram-Negative Multidrug-Resistant Bacteria among Patients Hospitalized in Two Centres in Ulaanbaatar, Mongolia.

Gram-negative multidrug-resistant organisms (GN-MDRO) producing β-lactamases (ESBL, plasmid-mediated AmpC β-lactamases and carbapenemases) are increasingly reported throughout Asia. The aim of this surveillance study was to determine the rate of bacterial colonization in patients from two hospitals in the Mongolian capital Ulaanbaatar. Rectal swabs were obtained from patients referred to the National Traumatology and Orthopaedics Research Centre (NTORC) or the Burn Treatment Centre (BTC) between July and September 2014, on admission and again after 14 days. Bacteria growing on selective chromogenic media (CHROMagar ESBL/KPC) were identified by MALDI-ToF MS. We performed susceptibility testing by disk diffusion and PCR (blaIMP-1, blaVIM, blaGES, blaNDM, blaKPC, blaOXA-48, blaGIM-1, blaOXA-23, blaOXA-24/40, blaOXA-51, blaOXA-58, blaOXA-143, blaOXA-235, blaCTX-M, blaSHV blaTEM and plasmid-mediated blaAmpC). Carbapenemase-producing isolates were additionally genotyped by PFGE and MLST. During the study period 985 patients in the NTORC and 65 patients in the BTC were screened on admission. The prevalence of GN-MDRO-carriage was 42.4% and 69.2% respectively (p<0.001). Due to the different medical specialities the two study populations differed significantly in age (p<0.029) and gender (p<0.001) with younger and more female patients in the burn centre (BTC). We did not observe a significant difference in colonization rate in the respective age groups in the total study population. In both centres most carriers were colonized with CTX-M-producing E. coli, followed by CTX-M-producing K. pneumoniae and CTX-M-producing E. cloacae. 158 patients from the NTORC were re-screened after 14 days of whom 99 had acquired a new GN-MDRO (p<0.001). Carbapenemases were detected in both centres in four OXA-58-producing A. baumannii isolates (ST642) and six VIM-2-producing P. aeruginosa isolates (ST235). This study shows a high overall prevalence of GN-MDRO in the study population and highlights the importance of routine surveillance, appropriate infection control practice and antibiotic prescribing policies to prevent further spread especially of carbapenemases.

Detection of presumptive Bacillus cereus in the Irish dairy farm environment

AbstractThe objective of the study was to isolate potentialBacillus cereussensu lato (B.cereus s.l.)from a range of farm environments. Samples of tap water, milking equipment rinse water, milk sediment filter, grass, soil and bulk tank milk were collected from 63 farms. In addition, milk liners were swabbed at the start and the end of milking, and swabs were taken from cows’ teats prior to milking. The samples were plated on mannitol egg yolk polymyxin agar (MYP) and presumptiveB. cereus s.l. colonies were isolated and stored in nutrient broth with 20% glycerol and frozen at -80 °C. These isolates were then plated on chromogenic medium (BACARA) and colonies identified as presumptiveB. cereus s.l. on this medium were subjected to 16S ribosomal RNA (rRNA) sequencing. Of the 507 isolates presumed to beB. cereus s.l. on the basis of growth on MYP, only 177 showed growth typical ofB. cereus s.l. on BACARA agar. The use of 16S rRNA sequencing to identify isolates that grew on BACARA confirmed that the majority of isolates belonged toB. cereus s.l. A total of 81 of the 98 isolates sequenced were tentatively identified as presumptiveB. cereus s.l. Pulsed-field gel electrophoresis was carried out on milk and soil isolates from seven farms that were identified as having presumptiveB. cereus s.l. No pulsotype was shared by isolates from soil and milk on the same farm. PresumptiveB. cereus s.l. was widely distributed within the dairy farm environment.

A nitrocefin-based amperometric assay for the rapid quantification of extended-spectrum β-lactamase-producing Escherichia coli in wastewaters

A sensitive and inexpensive amperometric assay based on the electrochemical detection of the β-lactamase activity using the nitrocefin as substrate was developed for the rapid and quantitative detection of extended spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) in urban wastewaters. The specific detection of ESBL-EC was achieved by culturing the filtered sample in a medium containing the cefotaxime supplemented or not with the potassium clavulanate inhibitor. This step was followed by the incubation of each subculture filtrate with the nitrocefin substrate which hydrolysis was monitored by amperometry using disposable carbon screen-printed sensors. Current intensities iCef and iClav correspond to the intensity of the anodic current measured (∼+ 0.2 V vs. Ag/AgCl) for the sample incubated with the cefotaxime without and with potassium clavulanate, respectively. The intensity value i = iCef – iClav was chosen as the analytical response. ESBL-EC calibration plots were established with artificially contaminated wastewater samples. This assay allowed the detection of ESBL-EC amounts as low as 10 cfu in treated effluents and 100 cfu in raw wastewaters with short time analysis of 5.5 h and 4.5 h, respectively. The amperometric method was applied to the analysis of 38 wastewater samples and the results were in good agreement with CFU counts on a selective chromogenic medium for 24 h. Owing to its rapidity, convenience, low-cost and portability, this assay is a promising tool to obtain quantitative data on antimicrobial-resistant E. coli in wastewater effluents. Furthermore, this assay might be used to improve wastewater treatment plant processes in order to minimize the release of antibiotic resistant bacteria into the aquatic environment.

Simultaneous Identification and Antimicrobial Susceptibility Testing of Multiple Uropathogens on a Microfluidic Chip with Paper-Supported Cell Culture Arrays.

A microfluidic chip was developed for one-step identification and antimicrobial susceptibility testing (AST) of multiple uropathogens. The polydimethylsiloxane (PDMS) microchip used had features of cell culture chamber arrays connected through a sample introduction channel. At the bottom of each chamber, a paper substrate preloaded with chromogenic media and antimicrobial agents was embedded. By integrating a hydrophobic membrane valve on the microchip, the urine sample can be equally distributed into and confined in individual chambers. The identification and AST assays on multiple uropathogens were performed by combining the spatial resolution of the cell culture arrays and the color resolution from the chromogenic reaction. The composite microbial testing assay was based on dynamic changes in color in a serial of chambers. The bacterial antimicrobial susceptibility was determined by the lowest concentration of an antimicrobial agent that is capable of inhibiting the chromogenic reaction. Using three common uropathogenic bacteria as test models, the developed microfluidic approach was demonstrated to be able to complete the multiple colorimetric assays in 15 h. The accuracy of the microchip method, in comparison with that of the conventional approach, showed a coincidence of 94.1%. Our data suggest this microfluidic approach will be a promising tool for simple and fast uropathogen testing in resource-limited settings.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species.

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 °C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

PERFORMANCE OF CHROM AGAR MEDIUM AND CONVENTIONAL METHODS FOR DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS.

Background:- Methicillin resistant Staphylococcus aureus (MRSA) are responsible for hospital and community acquired infections. There are many laboratory methods for detection of MRSA. Chromogenic media have been used for the last few years for the quick detection of MRSA. Objective:- Aim of this study was to compare the performance of conventional methods and chromogenic media for the detection of MRSA in a tertiary care hospital. Material and method: - 200 consecutive isolates of S. aureus confirmed by conventional methods, collected in a tertiary care hospital were used for this study. Cefoxitin and oxacillin disc diffusion test used as conventional methods and Chromogenic media i.e. oxacillin resistant screen agar base (ORSAB) was used for detection of methicillin resistant Staphylococcus aureus. All confirmed MRSA were checked by gold standard mecA base PCR method. Result: - Out of 200 isolates of Staphylococcus aureus, 50,52 and 47 strains were MRSA by Cefoxitin disc diffusion method, oxacillin disc diffusion method and oxacillin resistant screen agar base (ORSAB) method respectively. Specificity was 100%, 98.66%, 98.66% by Cefoxitin disc diffusion, oxacillin disc diffusion and ORSAB method respectively. Conclusion: - In conclusion, cefoxitin disc diffusion was the best for the phenotypic detection of MRSA because their sensitivity and specificity were better than oxacillin and ORSAB.

Rapid Detection of Enterobacter Sakazakii in milk Powder using amino modified chitosan immunomagnetic beads

Chitosan immunomagnetic beads (CIBs) were first prepared through converting hydroxyl groups of natural polymer material-chitosan into amino groups using epichlorohydrin and ethylenediamine as modification agent and then coupling with polyclonal antibodies of Enterobacter sakazakii using glutaraldehyde as cross-linking agent. The beads before coupling with antibodies were characterized by magnetic property measurement, FTIR, SEM and XRD technologies. In the assay a natural polysaccharide-chitosan, which has good biological and chemical properties such as non-toxicity, biocompatibility and high chemical reactivity was first used for synthesis of immunomagnetic beads. The detection method first established in this paper that combined the beads with chromogenic medium together to rapid detect E. sakazakii in milk powder could greatly improve the detection specificity and working efficiency. The beads exhibited a maximum capturing capacity of 1×106cfu/g with the detection sensitivity of 4cfu/g. The results demonstrate that the assay is a straightforward, specific and sensitive alternative for rapid detection of E.sakazakii in food matrix. The total analysis time was as little as about 25h, which greatly shorten the detection time. The method can provides new ideas not only to preparation technique of immunomagnetic beads but to imunne detection technique in food safety.