Hektoen Enteric Agar Research Articles

Bacterial species in biofilm cultivated from the end of the Seoul water distribution system.

To investigate changes in the bacterial population and the safety of the biofilm at the end of the drinking water distribution system in Seoul (Korea), selective media and bacterial community analyses were applied to a semi-pilot galvanized iron pipe (GIP) model. No total coliforms or faecal streptococci were detected on m-Endo or m-Enterococcus agar. No Salmonella spp. and Shigella spp. were detected on bismuth sulphite agar or Hektoen enteric agar, respectively. The latter two media detected coliforms, where m-Endo was negative. Biofilm formation started within 1 week (ca 104 CFU cm(-2)) and exceeded 105 CFU cm(-2) within 6 weeks. Although the fatty acid methyl ester analysis revealed dynamic changes in bacterial composition, Micrococcus, Bacillus, and Pseudomonas spp. were persistent members of the biofilm community. Micrococcus spp. was detected most frequently and in high numbers. Coliforms and Enterococcus species can be recovered from biofilms in water distribution systems. This study illustrates the role of biofilms in the chronic deterioration of the water-distribution system in Seoul (Korea).

Detection of Salmonella spp. in tropical seafood by polymerase chain reaction

The incidence of Salmonella spp. in tropical seafood was studied using standard microbiological techniques and polymerase chain reaction (PCR). Six of 20 finfish (30%), 4 of 20 clams (20%) and 1 of 20 shrimp (5%) were positive by culture techniques and by PCR. In a comparative study of different selective enrichment broths and selective plating media, more than one enrichment broth and selective agar were found to be necessary for efficient detection of Salmonella from seafood. Selenite cystine broth (SCB) was found to be more efficient compared to tetrathionate broth (TTB) while both bismuth sulfite agar (BSA) and hektoen enteric agar (HEA) were equally effective as selective plating media for fish. In the case of clams, HEA was found to be more effective. The presence of Salmonella spp. could be detected by PCR amplification of DNA extracted directly from the enrichment broths. In two cases, enrichment broths that were positive by PCR did not yield Salmonella by conventional methods.

Alafosfalin as a Selective Agent for Isolation of Salmonella from Clinical Samples

The selectivity of a range of culture media for the detection of Salmonella was assessed using 435 strains of gram-negative bacteria. These media showed limited ability to inhibit non-Salmonella strains found in stool samples. We report the evaluation of alafosfalin as a selective agent for isolation of Salmonella from stool samples. Susceptibility studies with this agent showed that non-typhi Salmonella strains were relatively resistant (mean MIC, 10.2 mg/liter) compared to many coliforms including Escherichia coli (mean MIC, 0.7 mg/liter). A chromogenic medium, ABC medium, was modified to incorporate alafosfalin and was compared with standard ABC medium and Hektoen enteric agar for the isolation of Salmonella from 1,000 stool samples. On direct culture, modified ABC medium showed higher recovery of Salmonella (53.6%) compared with either ABC medium (35.7%) or Hektoen enteric agar (48.2%). We conclude that alafosfalin is a useful selective agent for the isolation of Salmonella from stool samples.

Development of Methods for the Recovery of Escherichia coli O157:H7 and Salmonella from Beef Carcass Sponge Samples and Bovine Fecal and Hide Samples

Culture methods were developed for the concurrent recovery of Escherichia coli O157:H7 and Salmonella from bovine carcass, hide, and fecal samples. Several enrichment conditions were tested for the overall growth of pure cultures; tryptic soy broth for 2 h at 25°C and then for 6 h at 42°C was the protocol selected for use. Immunomagnetic separation (IMS) was incorporated for sensitivity and selectivity, along with a post-IMS enrichment for the recovery of Salmonella as recommended by the manufacturer. Selective agars for plating after IMS were chosen on the basis of ease of target colony identification. Sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and Rainbow agar supplemented with novobiocin and potassium tellurite were chosen for the recovery of E. coli O157:H7. Brilliant green agar with sulfadiazine and Hektoen enteric agar supplemented with novobiocin were selected for the recovery of Salmonella. The resulting methods were evaluated along with standard or previously used methods for the recovery of E. coli O157:H7 and Salmonella from bovine hide and fecal samples and carcass sponge samples. The Meats Research Unit (MRU) methods performed at least as well as the established methods, except that a secondary enrichment in tetrathionate (TT) broth prior to IMS was required for the optimal recovery of Salmonella from feces. Thus, the MRU and MRU-TT methods are effective in the recovery of both E. coli O157: H7 and Salmonella from a single bovine carcass, hide, or fecal sample.

Evaluation of culture media for enrichment and isolation of Shigella sonnei and S. flexneri

The performance of Gram-negative (GN) broth with (10 μg/ml) and without novobiocin, Shigella broth (SB) with 0.5 and 3.0 μg/ml novobiocin, all incubated at 37 °C (SB with 3.0 μg/ml novobiocin also at 42 °C) and buffered brilliant green bile glucose (EE) broth with 1.0 μg/ml novobiocin incubated at 37 and 42 °C were evaluated for resuscitation and growth of Shigella sonnei and S. flexneri (eight strains; unstressed, chill-stressed and acid-stressed) and non-shigellae (11 strains). GN broth with or without novobiocin supported significantly less growth of Shigella sp. No significant differences in growth of shigellae were obtained between the other culture media. Performance depended more on the Shigella strain used. None of the tested media were significantly superior for suppressing the competitive flora. Electivity and selectivity of MacConkey agar (MAC), tergitol-7 agar (T7), desoxycholate citrate agar (DCA), xylose lysine desoxycholate agar (XLD), Salmonella Shigella agar and Hektoen enteric agar (HEA) were determined by ecometric testing. HEA confirmed to be a high selective medium for both non-shigellae and stressed Shigella sp. Klebsiella sp., Enterobacter sp., Citrobacter sp., Salmonella sp. and the Escherichia strains can mask the presence of shigellae. In vitro competition experiments and experiments with artificially contaminated foods showed higher resistance of S. sonnei than S. flexneri towards the stress imposed by the food matrix and its indigenous flora. Reliable detection, however, of shigellae in foods with the current enrichment and isolation media was not achieved.

Improved Detection of Nontyphoid and Typhoid Salmonellae with Balanced Agar Formulations

A strategically balanced medium was developed for the improved detection of nontyphoid and typhoid salmonellae. Its balanced sugar (cellobiose, lactose, mannitol, and trehalose) and protein (beef extract and polypeptone peptone) formulation provided Salmonella with a selective growth advantage over non-Salmonella enteric organisms. The formulations promoted the production and detection of H2S production levels that otherwise might be missed with traditional agar formulations. In combination, these advantages contributed to increased sensitivity without the loss of specificity. In comparative studies using 86 samples of meat products (beef, pork, and chicken), the new media, Miller-Mallinson (MM) agar and xylose lysine tergitol (Niaproof) 4 agar, possessed significantly higher sensitivity (P < 0.001) and an improved specificity over bismuth sulfite, hektoen enteric, and xylose lysine desoxycholate agars. However, these samples did not contain nontyphoid salmonellae with weak to ultraweak H2S production characteristics. Modified formulations of MM agar were generally similar to bismuth sulfite and hektoen enteric agars in the identification of four of seven globally diverse strains of Salmonella serotype Typhi. Two of these seven strains were found to produce more readily identifiable black (H2S-positive) colonies on MM agar, whereas one of the seven was not readily detected by any of the media. The improved detection of nontyphoid and typhoid salmonellae attests to the sensitivity of MM agar and to its potentially broad utility in both clinical and food quality laboratories.

Comparação de Meios de Enriquecimento e de Plaqueamento Utilizados na Pesquisa de Salmonella em Carcaças de Frango e Fezes de Aves

Este trabalho foi desenvolvido para avaliar, comparativamente, os caldos de enriquecimento Rapapport-novobiocina (RVN), selenito-cistinanovobiocina (SCN) e tetrationato-novobiocina (TN) e os meios para plaqueamento ágar Hektoen (HE), ágar MacConkey (MC), ágar Salmonella-Shigella (SS), ágar verde brilhante (VB) e ágar xilose lisina desoxicolato (XLD), utilizados no isolamento de Salmonella em carcaças de frango e fezes de aves. O procedimento bacteriológico consistiu das etapas de pré-enriquecimento, enriquecimento em caldos seletivos, plaqueamento, testes bioquímicos presuntivos e confirmação sorológica com soros polivalentes anti antígenos somáticos e flagelares de Salmonella.. As fezes foram experimentalmente contaminadas com 8 sorotipos de Salmonella (Agona, Anatum, Enteritidis, Havana, Infantis, Owakam, Schwazengrund e Typhimurium) e a concentração final foi aproximadamente de 1,2 x 10² ufc/g. As fezes foram inoculadas nos caldos enriquecedores e a partir daí, seguiu-se o mesmo procedimento utilizado para as carcaças. Os resultados referentes às carcaças de frango não foram estatisticamente diferentes (p&gt;0,01) para os caldos e as placas. Todavia, verificou-se superioridade numérica em relação aos caldos SCN e TN sobre o caldo RVN, e em relação ao ágar XLD sobre os demais. Verificou-se também que com o emprego de dois caldos de enriquecimento e de dois meios para plaqueamento pode-se obter maior positividade. Quanto ao exame das fezes, o caldo TN mostrou-se superior aos demais (p&gt;0,01), não havendo diferença (p&gt;0,01) de resultados para os meios de plaqueamento. Os resultados sugerem que a utilização de mais de um meio de enriquecimento e de plaqueamento poderia aumentar as chances de isolamento de Salmonella.

Novel self-sampling culture method to monitor excretion of live, oral Shigella flexneri 2a vaccine SC602 during a community-based phase 1 trial

A culture technique for assessing the excretion of live enteric vaccines was developed and verified during an outpatient safety trial of the Shigella flexneri 2a SC602 vaccine. Preliminary studies showed that SC602 could be recovered on Hektoen enteric (HE) agar plates that had been inoculated with seeded stools in one quadrant, held for up to 48 hours, streaked for isolation, and incubated for 24 ± 6 hours. Recovery results on HE plates held at 4°C and 25°C were comparable; however, 4°C better inhibited overgrowth before streaking. To prepare for a community-based vaccine trial, volunteers were trained to self-sample fresh stool and to swabinoculate a single quadrant of HE agar. The trial began with 36 volunteers ingesting 2.5 × 104 CFU of SC602 in bicarbonate buffer. During the study, volunteers inoculated HE plates with fresh stool, stored the plates at 4°C, and delivered them to the laboratory within 48 hours. A microbiologist then streaked the HE for isolation, incubated the plates at 35°C ± 2°C for 24 ± 6 hours, and identified presumptive S. flexneri colonies by slide agglutination with poly-group B antiserum. The attenuating genetic signature of SC602 was confirmed on selected isolates with the polymerase chain reaction with two specific DNA primer sets. Vaccine was detected from 20% of volunteers on day 1, increasing to 86% by day 4, and all but one vaccinee had excreted SC602 at least once by day 7. The latest initial SC602 detection occurred on day 7, the longest excretion occurred in one vaccinee on day 33, and excretion throughout the trial was intermittent. The trial was terminated by ciprofloxacin treatment on day 35. Volunteer compliance with self-sampling and HE plating was excellent because of the convenience of the method, and the advantage of immediate “bedside” plating was evident in the high recovery rate of excreted vaccine. This method can be applied in other trials of live enteric vaccines that require accurate sampling of excreted organisms.

Evaluation of Immunomagnetic Separation and Plating Media for Recovery of Salmonella from Meat

Immunomagnetic separation (IMS) was compared with selective enrichment in selenite cystine (SC) broth for isolation of Salmonella from 86 artificially contaminated ground beef samples. Both Rambach agar (RA) and Hektoen enteric (HE) agar were used as selective plating media. The highest count of Salmonella colonies per plate was obtained after enrichment in SC broth and plating on RA (mean value: 111.1±58.1 CFU per plate); the lowest count was obtained after IMS and plating on HE agar (mean value: 65.4±36.6 CFU per plate). Salmonella in preenrichment was concentrated 1.7-fold by IMS and represented 31% of the microbial population captured by the beads, but nonspecific binding was high. As a result of the large numbers of competing bacteria, isolations on both RA and HE agar following IMS were quite difficult (mean value for Salmonella colonies: 79.9±42.7 CFU per plate; mean value for non-Salmonella colonies: 144.1±75.7 CFU per plate; ratio of Salmonella to non-Salmonella colonies: 0.8). This study indicates that SC broth is superior to IMS in the isolation of Salmonella from raw ground meat.

A novel chromogenic ester agar medium for detection of Salmonellae.

A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described. The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-[2-(4-octanoyloxy-3, 5-dimethoxyphenyl)-vinyl]-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14. 65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter-1. The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C8 fatty acid and a phenolic chromophore. In CSE agar, the ester is hydrolyzed by Salmonella spp. to yield a brightly colored phenol which remains tightly bound within colonies. After 24 h of incubation at 37 or 42 degreesC, colonies of typical Salmonella spp. were burgundy colored on a transparent yellow background, whereas non-Salmonella spp. were white, cream, yellow or transparent. CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars. The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82. 8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars. The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%). Strains of Citrobacter freundii and Proteus spp. giving false-positive reactions with other media gave a negative color reaction on CSE agar. CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997.

Growth of Salmonella enteritidis and S. pullorum on Hektoen agar and the expression of lipopolysaccharide or flagella

Growth of strains of Salmonella enteritidis and Salmonella pullorum on Hektoen agar has been reported to influence the expression of long-chain lipopolysaccharide and motility respectively. In this study we used a panel of strains of S. enteritidis and S. pullorum to investigate these phenomena. Culture on Hektoen agar did not cause rough strains of S. enteritidis to express long-chain lipopolysaccharide or strains of S. pullorum to become motile. It was concluded that growth of strains of S. enteritidis and S. pullorum on Hektoen agar would not normally affect the expression of somatic or flagellar antigens, and would not influence the interpretation of the Kauffman-White typing scheme.

Growth of Salmonella enteritidis and S. pullorum on Hektoen agar and the expression of lipopolysaccharide or flagella.

Growth of strains of Salmonella enteritidis and Salmonella pullorum on Hektoen agar has been reported to influence the expression of long-chain lipopolysaccharide and motility respectively. In this study we used a panel of strains of S. enteritidis and S. pullorum to investigate these phenomena. Culture on Hektoen agar did not cause rough strains of S. enteritidis to express long-chain lipopolysaccharide or strains of S. pullorum to become motile. It was concluded that growth of strains of S. enteritidis and S. pullorum on Hektoen agar would not normally affect the expression of somatic or flagellar antigens, and would not influence the interpretation of the Kauffman-White typing scheme.

Stool cultures for Shigella spp: Improved specificity by using MacConkey agar with xylose

A total of 678 stool specimens were cultured on four different agars: on xylose-lysine-desoxycholate agar (XLD), MacConkey agar (Mac), MacConkey agar supplemented with xylose (Mac-X), and Hektoen enteric agar (HE). Isolation rates for shigellae were 77% on HE, 86% on Mac and Mac-X, and 91% on XLD. The specificities of the media were 61% for Mac, 75% for HE, and 78% for XLD and Mac-X. After overnight incubation, Mac-X is much easier to read than XLD, which requires incubation for at least 22 hours. Based on these results and also on the practical aspect that incubation for 22-2l hours does not fit well in our schedule, we now use Mac-X whenever shigellae need to be looked for (i.e. mainly patients with recent travel to tropical countries). As compared to our previous procedure the workload in the laboratory could be reduced by about 20%.

Efficiency of different enrichment and isolation procedures for the detection of Salmonella serotypes in edible offal.

Rapid detection systems for Salmonella in foodstuffs are currently being developed. However, existing standards still call for application of traditional methods employing pre-enrichment followed by selective enrichment and isolation. The efficacy of various methods was tested using 264 chicken and lamb organ meats. Pre-enrichment was carried out in Tryptone Soy Broth (TSB) and enrichment in Tetrathionate Brilliant Green Broth (TTB) at 37 degrees C, Selenite Broth with Brilliant Green and Sulphapyridine at 37 degrees C and 43 degrees C, and Rappaport-Vassiliadis Broth (RV 10) at 42 degrees C. The isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen Enteric Agar (HEA) and Salmonella-Shigella Agar. Enrichment in RV/42 degrees C followed by isolation on BGA as recommended by ISO standard no. 6579 and enrichment in TTB/37 degrees C followed by isolation in HEA, no longer recommended by that standard, produced the best results. Low percentages of positive samples and difficulties in detecting Salmonella are the result of interference by competing organisms (Enterobacteriaceae) and the number of salmonellas present after enrichment. A total of 528 samples (TSB, eggs, lamb liver and chicken liver) were inoculated with Salm. enteritidis, Salm. kapemba and Salm. virchow, and the preceding experiment was repeated. All the TSB and egg samples tested positive, but the percentage of positive samples from the lamb and chicken liver was only 81-92%. Recovery of the salmonellas did not depend upon the method employed or the serotype inoculated but instead on interference by competing flora and the numbers of Salmonella present in the samples.

Comparison of three rapid methods for identification of Salmonella spp

A study was carried out to compare three rapid methods for detection of Salmonella spp. The fluorogenic MUCAP test (Biolife, Italy), the SM-ID agar test (bioMerieux, France) and the Rambach agar test (Merck, Germany) were used in this study to examine 103 strains (69 Salmonella strains and 34 non-Salmonella strains). Two conventional culture media, Hektoen and Leifson agars, were also included. The sensitivities of the MUCAP, SM-ID, Rambach and Hektoen agar tests for pure strains were 100, 93, 88 and 99%, respectively, and their specificities were 74, 97, 76 and 59%, respectively. A total of 100 stool samples from patients with acute diarrhoea was also tested and showed great discrepancy between the different methods. In agreement with other investigators, it was found that the discriminating capacity of Rambach and SM-ID as primary plating media was very restricted. The MUCAP test was very sensitive, rapid and easy to perform but not very specific. In view of these results, it is essential to combine different methods for the accurate and reliable detection of Salmonella strains.

Stool culture: are you getting value for money?

To assess the best medium for primary isolation of enteric pathogens; to determine the need for a second primary culture medium; and to gather information on stool culture media used in 20 other randomly selected laboratories. Specimens were cultured on desoxycholate citrate agar (DCA), Hektoen enteric agar (Hektoen), and xylose lysine deoxycholate agar (XLD). Non-lactose fermenters were screened with Rapidec Z (bio-Mérieux) and identified with API 10S (bio-Mérieux) where appropriate. Shigellas were identified with API 20E (bio-Mérieux) and serology, and salmonellas biochemically and by serology. A telephone survey was carried out to enquire into different culture practices and whether they had been evaluated for cost effectiveness. The isolation rate of enteric pathogens on primary stool culture media was 97% on DCA, 88% on XLD, and 76% on Hektoen. Seventeen of 18 shigellas grew on DCA, 13 of 18 on XLD, and 14 of 18 on Hektoen. DCA missed one Salmonella, XLD three, and Hektoen 13. XLD and Hektoen both missed Yersinia enterocolitica. The telephone survey revealed a diverse range of both primary and subculture plates. There was little evidence of evaluation of stool media, but firm personal convictions concerning the advantages and disadvantages of each type of medium at each stage of culture. DCA performed best and was the most cost effective of the three media. Neither XLD nor Hektoen were satisfactory as primary culture media because they grew fewer pathogens than DCA.

Evaluation of two new chromogenic media for detection ofSalmonella in stools

A clinical collaborative study was conducted to compare two new chromogenic agar media, Rambach agar and the Salmonella Detection and Identification Medium (SMID) (bioMérieux, France), with two conventional media, Salmonella-Shigella agar and Hektoen agar. Thirty-nine Salmonella strains involving 14 serotypes were isolated from 1,454 stool specimens. After enrichment in a selective broth, 100% sensitivity was obtained with each medium. The SMID and Rambach agars are considerably more specific than the conventional media. Although SMID agar detects all Salmonella serotypes, it is not as specific as Rambach agar, which requires a complementary test (C8 esterase test) to detect all serotypes.

Growth of Salmonella spp. in Cantaloupe, Watermelon, and Honeydew Melons

The ability of Salmonella spp. to grow on the interior tissues of cantaloupe, watermelon, and honeydew melons was investigated. Pieces of rind-free melons (pH 5.90–6.67) and tryptic soy broth (TSB, pH 5.90) were inoculated with a mixed culture (approximately 100 CFU/g or ml) containing equal proportions of five species of Salmonella (S. anatum, S. Chester, S. havana, S. poona, and S. senftenberg). Inoculated melon pieces and TSB were incubated for 24 h at 5 or 23°C. Viable populations of salmonellae were determined by surface plating test portions on Hektoen enteric agar. Results indicated that Salmonella growth was rapid and prolific on the melons and in TSB at 23°C incubation. Final populations on watermelons were approximately 1.0 log10 greater than populations on cantaloupe and honeydew and in TSB. Although viable Salmonella populations on melons and in TSB did not increase during the 24-h incubation at 5°C, little or no decrease in viable populations was observed.

USE of an ELECTRONIC HGMF INTERPRETER SYSTEM FOR ENUMERATION of NALIDIXIC ACID RESISTANT SALMONELLAE IN CHICKEN CAECA

An electronic counting system using hydrophobic grid‐membrane filters (HGMF) and the HGMF Interpreter was evaluated for its usefulness in enumerating nalidixic acid resistant Salmonella in frozen chicken caeca. Salmonella recovery was equivalent on both Hektoen Enteric and EF‐18 agars. However, the color of the Salmonella growth on EF‐18 agar was more easily differentiated by the HGMF Interpreter electronic counting system. the study showed that a 4 h resuscitation on a nonselective medium was required in order to maximize the subsequent recovery on Hektoen Enteric agar, though not on EF‐18 agar. Using the EF‐18 agar as the Salmonella selective medium, a method was established that recorded counts of nalidixic acid resistant Salmonella rapidly and easily in electronic data files, for subsequent retrieval and manipulation.

Salmonella detection in marine waters using a short standard method

A study was undertaken to compare two enrichment broths and to test a new plate medium, Rambach agar, for their suitability to detect Salmonella from marine recreational waters. The study shows that under conditions of low levels of fecal contamination, Salmonella spp was isolated more efficiently using NR10(10)/43 broth. While at higher levels of fecal contamination NR10/43 broth effectively limits the growth of accompanying flora. For the highest level of fecal contamination Rambach agar was less selective than Hektoen enteric agar but Rambach agar was shown to be better than Hektoen agar for the recovery of Salmonella strains from low contaminated waters. On Rambach agar the distinct color of salmonellas, seen in red, enhanced correct identification. Biochemical and serological confirmation of Salmonella isolates was performed using the API Z System and Oxoid Salmonella Rapid Latext test. The combination of enrichment broths (24 h at 42°C), isolation (24 h at 37°C) and identification systems described above (2 h or I min) allowed the detection of Salmonella from marine waters, with low or high levels of fecal contamination, in 48 h.