DHL Agar Research Articles

Prevalence of Antibiotic Resistant Bacteria in Ready to eat Food Sold by Street Vendors in Bangladesh

Bangladesh has a vibrant street food culture, with easily available ready-to-eat (RTE) foods. In this study, a total of 221 isolates were obtained from 141 Bangladeshi RTE food samples, such as fried, non-fried, and cooked foods; egg-, milk-, cereal-, and cream-based foods; pickles/achar; fruit; and RTE leaves, through culture on trypticase soy (for aerobic plate count), mannitol salt (MS for Staphylococcaceae), deoxycholate-hydrogen sulfide-lactose (DHL, for Enterobacteriaceae), and NaCl glycine Kim Goepfert agar (NGKG, for Bacillus cereus-like bacteria) agar plates. The aerobic plate counts ranged from undetectable to 8.5 log CFU/g. After enrichment with peptone water, contaminated bacteria detected on DHL, MS, and NGKG agar plates, from 77, 110, and 77 samples, respectively. Twenty out of 111 isolates on DHL agar, and 17 of 32 isolates on NGKG agar clearly showed resistance against three or more drugs. Through 16S ribosomal DNA sequencing analysis, six selected isolates from DHL agar were identified as Pseudomonas nitroreducens, Citrobacter braakii, Klebsiella pneumoniae subsp. pneumoniae, and Serratia marcescens. One selected isolate from NGKG agar was identified as Bacillus cereus-like bacteria. The results suggest that additional safety measures and regulations are necessary to ensure the quality and safety of the RTE foods in Bangladesh.

Selection of broad‐spectrum cephalosporin‐resistant Escherichia coli in the feces of healthy dogs after administration of first‐generation cephalosporins

ABSTRACTAlthough antimicrobial products are essential for treating diseases caused by bacteria, antimicrobial treatment selects for antimicrobial‐resistant (AMR) bacteria. The aim of this study was to determine the effects of administration of first‐generation cephalosporins on development of resistant Escherichia coli in dog feces. The proportions of cephalexin (LEX)‐resistant E. coli in fecal samples of three healthy dogs treated i.v. with cefazolin before castration and then orally with LEX for 3 days post‐operation (PO) were examined using DHL agar with or without LEX (50 µg/mL). LEX‐resistant E. coli were found within 3 days PO, accounted for 100% of all identified E. coli 3–5 days PO in all dogs, and were predominantly found until 12 days PO. LEX‐resistant E. coli isolates on DHL agar containing LEX were subjected to antimicrobial susceptibility testing, pulsed‐field gel electrophoresis (PFGE) genotyping, β‐lactamase typing and plasmid profiling. All isolates tested exhibited cefotaxime (CTX) resistance (CTX minimal inhibitory concentration ≥4 µg/mL). Seven PFGE profiles were classified into five groups and three β‐lactamase combinations (blaCMY‐4‐blaTEM‐1, blaTEM‐1‐blaCTX‐M‐15 and blaTEM‐1‐blaCTX‐M‐15‐blaCMY‐4). All isolates exhibited identical PFGE profiles in all dogs on four days PO and subsequently showed divergent PFGE profiles. Our results indicate there are two selection periods for AMR bacteria resulting from the use of antimicrobials. Thus, continuing hygiene practices are necessary to prevent AMR bacteria transfer via dog feces after antimicrobial administration.

腸管出血性大腸菌感染症患者糞便中の原因菌量と 酵素基質培地クロモアガーSTEC の有用性

We report a case of enterohemorrhagic Escherichia coli (EHEC) infection in which EHEC was not detected by culture on DHL agar medium. The proportion of EHEC bacterial count to enterobacterial count in feces was 1.7%, and the detection probability by 5-colony angling was low (8.1%). The probability of angling detection using CHROMagar STEC, a chromogenic medium for detecting EHEC, was high (100%). An additional and collection test was done using E. coli bacterial solutions to which two main sera groups--O157 and O26 were added. The maximum detectable level in the bacterial solution with O157 was 10(3)-10(4) CFU/mL in DHL and 10(2) CFU/mL in CHROMagar STEC. Bacterial solution levels with O26 were 10(3) CFU/mL in DHL and 10(2) CFU/mL in CHROMagar STEC. Assuming that the EHEC bacterial amount in feces of those with EHEC infection is low, we speculated that CHROMagar STEC may be useful as on EHEC screening medium.

Detection of non-typhoid Salmonella infection by citrus and citrus extracts in Lao PDR

Objective To know the current state of non-typhoid Salmonella infection in Laos. To examine the usefulness of new screening methods for Salmonella using citrus. Methods Non-typhoid Salmonella infection of person in Lao PDR was studied in this research (2004-2009). The site was Vientiane capital city in 2004. Research from rural villages locating suburb of Vientiane during 2005-2008 was carried out. Rural villages in Attapu province where ethnic minorities were living was searched for this study in 2009. During this research, to detect Salmonella strain, a new method using citrus and citrus extract named MY phenomenon that observing black ring (MIDO ring) on DHL agar was tried. The slice lemon and lime were used for this trial in 2004. After 2005, disk of ascorbic acid and citric acid were used for the device instead of citrus fruits itself. Results During this research, 65 of 272 human samples (23.9%) were infected with non-typhoid Salmonella. Conclusions During this study, the method using citrus and citrus extracts was accepted for the detection of Salmonella. This study shows that with citrus and citrus extract, detection of Salmonella is possible using only DHL media. Results suggest that infectious rate of non-typhoid Salmonella was high.

Dextran sulfate sodium and 2,4,6-trinitrobenzene sulfonic acid induce lipid peroxidation by the proliferation of intestinal gram-negative bacteria in mice.

AbstrectBackgroundTo understand whether TLR-4-linked NF-kB activation negatively correlates with lipid peroxidation in colitic animal models, we caused colitis by the treatment with dextran sulfate sodium (DSS) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) to C3H/HeJ (TLR-4-defective) and C3H/HeN (wild type) mice, investigated inflammatory markers, lipid peroxidation, proinflammatory cytokines and TLR-4-linked NF-κB activation, in colon and intestinal bacterial composition in vivo.MethodsOrally administered DSS and intrarectally injected TNBS all caused severe inflammation, manifested by shortened colons in both mice. These agents increased intestinal myeloperoxidase activity and the expression of the proinflammatory cytokines, IL-1β, TNF-α and IL-6, in the colon.ResultsDSS and TNBS induced the protein expression of TLR-4 and activated transcription factor NF-κB. However, these colitic agents did not express TLR-4 in C3H/HeJ mice. Of proinflammatory cytokines, IL-1β was most potently expressed in C3H/HeN mice. IL-1β potently induced NF-κB activation in CaCo-2 cells, but did not induce TLR-4 expression. DSS and TNBS increased lipid peroxide (malondialdehyde) and 4-hydroxy-2-nonenal content in the colon, but reduced glutathione content and superoxide dismutase and catalase activities. These colitic inducers increased the number of Enterobacteriaceae grown in DHL agar plates in both mice, although the number of anaerobes and bifidobacteria grown in GAM and BL agar plates was reduced. E. coli, K. pneumoniae and Proteus mirabilis isolated in DHL agar plates increased lipid peroxidation in liposomes prepared by L-α-phosphatidylcholine, but B. animalis and B. cholerium isolated from BL agar plates inhibited it.DiscussionThese findings suggest that DSS and TNBS may cause colitis by inducing lipid peroxidation and enterobacterial proliferation, which may deteriorate the colitis by regulating proinflammatory cytokines via TLR-4-linked NF-κB activation pathway.

バイダスを用いたサルモネラ迅速検出法の検討

Both the incidence and number of patients with food-borne diseases caused by Salmonellastill rank very high among total food-borne diseases, although both have been decreasing in recent years. The need for a simple and rapid alternative method of Salmonellatesting is very high in the food industry because the conventional culture method includes two enrichment procedures and takes more than five days to isolate and identify a typical colony of Salmonella from samples. VIDAS is an enzyme-linked fluorescent assay (ELFA) system that consists of a fully automated device and its reagents. In this study, we compared two protocols of the VIDAS method with the conventional culture method for detecting Salmonella using 40 food samples. Performances of protocols “ VIDAS ICS + plate ”and “ VIDAS SLM AFNOR validated method” both showed higher sensitivity than the conventional culture method. These protocols were also excellent in terms of reduced time-toresults in addition to showing higher sensitivity. We also compared SMID 2, a new chromogenic agar plate for selective isolation of Salmonella, with DHL agar, a traditional agar plate for Salmonella testing. On SMID 2, typical colonies of Salmonella were much easier to identify (mainly by its characteristic color), and the frequency of appearance of typical colonies by bacteria other than Salmonella was much lower, although the Salmonella recovery rate did not differ between the two media.

Identification of Factors Involved in Recovery of Heat-Injured Salmonella Enteritidis

Proteins and genes involved in the recovery of heat-injured Salmonella Enteritidis were investigated. Salmonella Enter-itidis cells cultured overnight in tryptic soy broth (TSB; nonselective medium) were suspended in citric acid–disodium hydrogen phosphate buffer (pH 6). After heat treatment at 55°C for 15 min, the culturable counts measured by tryptic soy agar (TSA; nonselective medium) decreased from 108 to 107 CFU/ml. On the other hand, culturable counts measured by desoxycholate–hydrogen sulfite–lactose (DHL) agar (selective medium) were decreased from 108 to 104 CFU/ml by the same treatment. The results suggest that 99.9% of Salmonella Enteritidis detected on TSA were injured but recoverable. When injured Salmonella Enteritidis was incubated in TSB, the culturable count measured by TSA did not increase for 2 h, whereas that by DHL agar increased after incubation for 30 min. After incubation for 2 h, the culturable count measured by DHL agar reached a similar level with that by TSA, indicating that Salmonella Enteritidis had recovered. The two-dimensional polyacrylamide gel electrophoresis analysis revealed that elongation factor G (FusA) and pyruvate kinase (PykF) specifically increased in the cells just after heat treatment and in the recovery cells. The levels of transcription of 86 stress-inducible genes were also investigated by reverse transcription PCR. Nineteen heat-inducible (clpB, clpX, degP, dnaJ, fkpA, ftsJ, gapA, hflB, hslJ, hslU, hslV, htpG, htrA, lon, mopA, mopB, mreB, rpoE, and ppiD), and 12 oxidative-stress and DNA damage–inducible (ahpC, ahpF, fldB, fur, grxA, dinF, katG, mutM, recA, soxR, trxC, and zwf) genes were transcribed extensively during recovery in TSB. The results obtained in this study will be used to develop the media or culture conditions that will promote recovery for the detection of food poisoning bacteria, including injured cells from food products.

시유의 2차오염과 저장가능기간을 결정하기 위한 Resazurin 환원시간검사

The selective media including NPC agar, DHL agar, MacConkey agar, and Cetrimide desoxycholate agar were compared to determine selectivity for the growth of bacteria. Cetrimide desoxycholate agar was better than NPC agar, DHL agar, and MacConkey agar for the growth of psychrotrophic gram-negatve bacteria in- cluding Pseudomonas. and for the inhibition of gram positive bacteria. The specificity of resazurin reduction time test was investigated to determine post-pasteurization contamination of market milk. Equal volume of Cetrimide desoxycholate broth was added to market milk, which was then incubated at 21 for 18 hours. ℃ The growth of bacteria in the incubated milk was detected in resazurin reduction time test. The results in re- sazurin reduction time test and total bacteria number count of market milk after storage at 7 were relatively ℃ correlated each other. Pseudomonas was isolated most frequently from the market milk stored at 7 for 10 ℃ days, and Acinetobacter and Aeromonas followed. Acinetobacter, Pseudomonas and Enterobacter were fre- quently isolated from the mixture of market milk and Cetrimide desoxycholate broth incubated at 21 for 18 ℃

Detection of Verocytotoxin-Producing Escherichia coli O157 by Polymerase Chain Reaction Assay After Immunomagnetic Separation-Plating.

For detection of verocytotoxin-producing Escherichia coil (VTEC) O157 in foods and feces, colony isolation on selective agar after immunomagnetic separation (IMS) was followed by polymerase chain reaction (PCR) assay. It was shown that Rainbow agar O157 could recover E. coli O157 more sensitively than other selective media such as Desoxycholate agar, DHL agar, SIB agar, CT-SMAC agar, BCM O157, Chrom agar O157 and Chrom agar E. coli. Also, the PCR assay of the typical colonies developed on Rainbow agar O157 was able to detect VTEC O157 in sample suspensions with more than 101 cfu/ml.

The Magnetic Immuno Polymerase Chain Reaction Assay for Detection of Salmonella from Food and Fecal Samples.

The present study was performed to isolate Salmonella by plating the bead-bacteria complexes on DHL agar after immunomagnetic separation (IMS) from food and fecal samples contaminated artificially at 1 to 103 cfu/ml, and then to detect Salmonella by polymerase chain reaction (PCR) assay. With IMS, it was possible to collect Salmonella from the samples at 1 cfu /ml. Even if colonies other than Salmonella developed on the selective agar plate, only Salmonella was detected by the PCR assay using two pairs of primers for the Salmonella invA and stn genes. Also, Salmonella was completely detected within 24 h from eggs contaminated naturally with Salmonella O9 and from diarrheal stool samples of patients with Salmonella O9 or O16. This combination of IMS and PCR seems to be a useful method for rapid and sensitive detection of Salmonella in food products and fecal specimens.

Biochemical Properties and Colony Formation Patterns of Salmonella-Like Strains.

Five bacterial strains were isolated from food samples, and were not easily distinguished from Salmonella via conventional methods. These bacterial strains wereidentified as Citrobacter spp. from their biochemical properties. Despite this, their characteristics after being cultured resembled those of Salmonella, with similar kinds of colony formation patterns on XLD, DHL and TSI agar. Since these salmonella-like strains grew at a rate and produced colonies with black centers characteristic of Salmonella, the selective detection of Salmonella in the presence of these salmonella-like strains was impossible, based on conventional methods.

Effects of Disinfectants and Spray Washing on Salmonella Enteritidis Contamination on the Shell Surface of Eggs

The effects of disinfectants and antibacterial chemicals used in food production on Salmonella Enteritidis contamination on the shell surface of eggs were examined from the viewpoint of prevention of Salmonella foodborne disease due to intact eggs and egg products (whole egg, eggwhite, yolk, sugared yolk, salted yolk, etc.).Eggs artificially contaminated with S. Enteritidis in various ways were immersed in a solution of sodium hypochlorite, organic acid (fumaric acid or acetic acid) or trisodium phosphate for 2 to 20min. After drying, these eggs were stored at 8 or 25°C for 14 days. S. Enteritidis on the shell surface was evaluated by direct plating on DHL agar (direct method) and by enrichment with EEM broth (enrichment method) at intervals of 0 (examined immediately), 1, 3, 7 and 14 days.The number of eggs contaminated with S. Enteritidis was decreased effectively by storage for 7 to 14 days at 8°C or 25°C after immersion in 5-15% trisodium phosphate solution. Eighty to one hundred percent of eggs examined were free from S. Enteritidis by the direct method, whereas 65-87% of eggs were uncontaminated according to the enrichment method. The number of S. Enteritidis on the shell surface of eggs artificially contaminated with the organism in 2% whole eggs and 1% gelatin was decreased effectively by spray washing with fresh water after immersion in 1-2% trisodium phosphate solution.From these results, trisodium phosphate may be effective for removing Salmonella from the shell surface of eggs during washing of eggs in grading and packing centers and in egg-product plants.

β-glucuronidase活性を利用した散発性下痢症患者からの下痢原性大腸菌の検出

To compare the isolating efficiency of diarrhea-causing Escherichia coli between Fluorocult agar plates, which reveal the beta-glucuronidase activity of E. coli, and a combination of SS and DHL agar plates, a total of 330 fecal specimens collected from outpatients were examined. Diarrhea-causing E. coli, identified by serological and toxigenic characters, were demonstrated in 52 samples. Among these specimens, 35 samples tested were positive on the Fluorocult agar plates, and 26 samples on the combination of SS and DHL agar plates. However, only 10 samples were positive on both the Fluorocult agar plates and the combination of SS and DHL agar plates. Thus, using Fluorocult agar plates for the isolation of diarrhea-causing E. coli in addition to the conventional SS and DHL agar plates will improve isolating efficacy.

沿岸二枚貝の細菌相

Bacterial flora in 6 species of bivalves, Tapes philippinarum, Mactra veneriformis, Mitylis coruscus, Crassostrea gigas, Phacosoma japonicum and Scapharca broughtonii, sediment and seawater in Tokyo bay were investigated by using PYBG, 1/20 PYBG, N-PYBG, GAM, DHL, LBS and PEA agar media under aerobic and anaerobic conditions. Different values of colony counts were observed on 7 different media. Major aerobic bacteria of bivalves were composed of Vibrio-Aeromonas, Pseudomonas, Enterobacteriaceae, Moraxella and Micrococcus. Anaerobic bacteria belonged to Bacteroidaceae and clostridia, which were minor components. Bacterial components of sediment and seawater were similar to those of bivalves. These results suggest that bacterial flora of bivalves are well influenced by environmental flora.

Evaluation of some cold enrichment and isolation media for the recovery of Yersinia enterocolitica

An evaluation of several cold enrichment media for Yersinia enterocolitica showed that the enrichment quotient achieved after 3 weeks at 4 degrees C was highly dependent on the initial cell concentration and the medium used. The latter should be of high nutritional value, in order to allow sufficient growth of Yersinia enterocolitica at a low temperature. Enrichment in typtone--soya broth yielded better results than in--frequently used--phosphate buffer, pH 7.6. While comparing isolation media for Yersinia enterocoliica to be used after cold enrichment, DHL agar was most satisfactory: after 20 h incubation at 29 degrees C, colonies of Yersinia enterocolitica are easily distinguishable and the organisms fully recovered. An urea medium, containing novobiocin as selective agent, also yielded good results. It must be stressed that only human strains of serotypes 0:3 and 0:9 of Yersinia enterocolitica were studied.

Gnotobiotic, SPFならびにConventional ICRマウスの糞便菌そうの検索

Fecal flora was examined in six groups of ICR mice which were reared in bacteriologically different environments. As shown in Table 1, gnotobiotic mice (Groups I & II) harbored only enteric bacteria, streptococci, staphylococci and lactobacilli. Bacteroides and yeasts appeared in two groups of SPF mice (Groups III & IV) which were derived from the mice of Group I. SPF mice of Group V, which were kept in an SPF room for four years, and conventional mice (Group VI), which were derived from the mice of Group V, harbored enteric bacteria, streptococci, staphylococci, lactobacilli, bacteroides, yeasts, aerobic spore formers, clostridia and pseudomonads.Subsequently, the organisms grown on DHL, SF, mannitol-salt and LBS agar plates were divided into several genera, species or biotypes (Table 2) . The organisms on DHL agar plates from gnotobiotic mice showed homologous properties and were classified in genus Proteus. On the other hand, the organisms consisting of Proteus and Klebsiella were isolated from SPF mice of Groups III and IV. SPF mice of Group V harbored Citrobacter, Escherichia and Klebsiella, while conventional mice harbored Escherichia including hydrogen-sulphide producing E. coli, Proteus and Citrobacter. As can be seen from Table 2, the same results as above were observed using SF, mannitol-salt and LBS agar plates, i. e, the number of species or types harbored increased when the gnotobiotic mice were transferred from isolator to SPF room or the SPF mice from SPF room to conventional room.