Comparison of enrichment broths for isolation of Campylobacter jejuni

Growth of Campylobacter jejuni was compared in enrichment broths of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) and Park et al. (C. E. Park, Z. K. Stankiewicz, J. Lovett, and J. Hunt, Can. J. Microbiol. 27:841-842, 1981), as modified by Lovett et al. (J. Lovett, D. W. Francis, and J. M. Hunt, Appl. Environ. Microbiol. 46:459-462, 1983). Inoculated foods used were cream-pudding types, which may be cross-contaminated by improper handling, improper storage of meats prepared simultaneously, or the use of raw milk as an ingredient. Both broths adequately supported growth.     

Comparison of enrichment broths for isolation of Campylobacter jejuni.

Growth of Campylobacter jejuni was compared in enrichment broths of Doyle and Roman (Appl. Environ. Microbiol. 43:1343-1353, 1982) and Park et al. (C. E. Park, Z. K. Stankiewicz, J. Lovett, and J. Hunt, Can. J. Microbiol. 27:841-842, 1981), as modified by Lovett et al. (J. Lovett, D. W. Francis, and J. M. Hunt, Appl. Environ. Microbiol. 46:459-462, 1983). Inoculated foods used were cream-pudding types, which may be cross-contaminated by improper handling, improper storage of meats prepared simultaneously, or the use of raw milk as an ingredient. Both broths adequately supported growth.     

A comparison of two culture media for the recovery of thermophilic campylobacters in broiler farm samples

A study to evaluate the performance of two different brands of media (Oxoid, Basingstoke, UK, and Mast Diagnostics, Merseyside, UK) for the isolation of thermophilic campylobacters from a range of broiler farm samples was undertaken. Oxoid media performed significantly better than the Mast formulations with overall Campylobacter recovery rates of 46% and 30.5%, respectively, observed from 213 samples tested (p< or =0.05). Consistently higher recoveries of campylobacters were observed from all samples when the results using both types of media were combined.     

Comparison of some decontamination methods and growth media for isolation of mycobacteria from northern brook waters

Two decontamination methods and five media were compared for the isolation of mycobacteria from brook waters of different physical, chemical and bacteriological characteristics. The decontaminants used were: 0.7 mol 1^-1 NaOH followed by 50 g 1^-1 oxalic acid and 0.9 mol 1^-1 H₂SO₄ combined with 0.5 g 1^-1 cycloheximide. The media compared were: Mycobacteria 7H11 agar with OADC enrichment (pH 6.6), glycerol egg (pH 6.5 and 5.5), and pyruvate egg (pH 6.5 and 5.5). All media contained cycloheximide, 0.5 g 1^-1. The NaOH—oxalic acid method generally resulted in lower contamination and higher isolation of mycobacteria than the H₂SO₄-cycloheximide method. With the NaOH—oxalic acid method, all five media were equal in positivity rates but contamination was a problem on Mycobacteria 7H11 agar. Of the four egg media tested, the highest positivity rate (92% of the samples) was obtained on the pyruvate modification (pH 6.5), and the highest mean colony count of mycobacteria (900 cfu 1^-1) on the glycerol modification (pH 6.5). Characteristics of water and sampling site had similar effects on the isolation frequencies of mycobacteria obtained by different combinations.     

Comparison of three enrichment media for the isolation of Campylobacter spp. from foods

AIM: This study compared the performance of three Campylobacter enrichment broths: Bolton broth (BB), Campylobacter Enrichment broth (CEB) and Preston broth (PB). METHODS AND RESULTS: Pure cultures of target and competitor organisms, and naturally-contaminated food samples, were used to establish the performance of these media. In pure culture the PB supported the growth of the greatest number of strains of Campylobacter spp. but failed to inhibit some competitor organisms. The CEB showed the opposite result, inhibiting all 15 competitor organisms used but failing to support the growth of five Campylobacter strains. By comparison, BB showed the best compromise between inhibition of competitors and growth of Campylobacter. CONCLUSIONS: Plates inoculated with BB and CEB food enrichments resulted in more Campylobacter growth than those inoculated with PB, which supported significantly less typical growth (P < or = 0.001). The most common competitor organism isolated from PB was Escherichia coli, and Pseudomonas spp. were frequently isolated from BB and CEB. Both BB and CEB were better than PB for the isolation of Campylobacter from naturally-contaminated foods, although BB yielded more confirmed Campylobacter growth than CEB. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted differences in performance of media used to isolate Campylobacter spp. from foods.     

Eight enrichment broths for the isolation of Campylobacter jejuni from inoculated suspensions and ground pork

Aims:  The efficiency of eight enrichment broths for the selective isolation of Campylobacter jejuni was compared to identify an optimal enrichment broth.
Methods and Results:  Brucella-FBP, Preston, Doyle and Roman, modified CCD (mCCD), Park and Sanders, Bolton, Hunt and Radle and Hunt broths were compared for their recovery of (i) Camp. jejuni in suspension, (ii) Camp. jejuni from inoculated ground pork, (iii) heat-injured Camp. jejuni (55°C for 20 min) in suspension and (iv) heat-injured Camp. jejuni from inoculated ground pork. Hunt broth and Bolton broth showed the highest and most rapid enrichment efficacy for the cell suspensions and ground pork, respectively. Preston, Park and Sanders and mCCD broths had relatively high enrichment efficiencies, while Brucella-FBP broth was significantly inferior to the other broths ( P  < 0·05).
Conclusions:  Cell recovery from the eight enrichment broths was dependent on the sample type and the state of the cells. The use of the appropriate broth is important for the rapid and efficacious enrichment of Camp. jejuni . In particular, heat-injured Camp. jejuni require a longer cultivation time and a suitable enrichment broth.
Significance and Impact of the Study:  The results from the present study provide information for selecting the most appropriate enrichment broth for Camp. jejuni and may contribute to improved detection methods for the organism.     

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°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 tryptone—soya broth yielded better results than in—frequently used—phosphate buffer, pH 7.6.While comparing isolation media for Yersinia enterocolitica to be used after cold enrichment, DHL agar was most satisfactory: after 20 h incubation at 29°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.     

Comparison of sampling techniques and different media for the enrichment and isolation of cellulolytic organisms from biogas fermenters

Biogas plants achieve its highest yield on plant biomass only with the most efficient hydrolysis of cellulose. This is driven by highly specialized hydrolytic microorganisms, which we have analyzed by investigating enrichment strategies for the isolation of cellulolytic bacteria out of a lab-scale biogas fermenter. We compared three different cultivation media as well as two different inoculation materials: Enrichment on filter paper in nylon bags (in sacco) or raw digestate. Next generation sequencing of the V3/V4 region of the bacterial 16S rRNA of metagenomic DNA from six different enrichment cultures, each in biological triplicates, revealed an average richness of 48 different OTU’s with an average evenness of 0.3 in each sample. β-Diversity of the bacterial community revealed significant differences between the two sampling techniques or the different media used. The isolation attempt of single cellulolytic organisms resulted in several clonal pure cultures. Regardless which medium or inoculation material, well-known cellulolytic key players such as Clostridium cellulosi, Herbinix hemicellulosilytica and Hungateiclostridium thermocellum were among the isolates. The inoculation material as well as the cultivation conditions are crucial to cultivate the representative cellulolytic organisms. Taking raw digestate as inoculation material and using the same material, filtered and sterilized, for supplementing media allowed to imitate the natural habitat. Pre-enrichment of cellulolytic organisms directly in their natural habitat led to significant advantages concerning high diversity and high abundance of unknown cellulolytic organisms, which is a key factor for the isolation of hitherto unknown species.     

Determination of the optimal culture conditions for detecting thermophilic campylobacters in environmental water

This study evaluated alternative protocols for culturing thermophilic campylobacters in environmental water. All samples were filtered through a sterile 0.45 μm pore-size membrane, which was then incubated in Preston enrichment broth. Four variables were compared: water sample volume (2000 mL vs. 500 mL), enrichment broth volume (25 mL vs. 100 mL), enrichment incubation duration (24 h vs. 48 h), and number of enrichment passages (one vs. two). In addition, DNA extracts were prepared from all final broths and analyzed using three rRNA PCR assays. River water was collected at 3 sampling sites weekly for 9 weeks. Among these 27 collections, 25 (93%) yielded Campylobacter spp. under at least one of the 16 culture conditions. By univariate analysis, yields were significantly better for the 2000 mL sample volume (68.5% vs. 43.0%, p < 0.0001) and the 25 mL enrichment broth volume (64.5% vs. 47.0%, p < 0.0004). Neither of the enrichment period had a significant effect, although there was a trend in favor of 48 h incubation (59.5% vs. 52.0%, p = 0.13). The three PCR methods gave concordant results for 66 (33%) of the culture-negative samples and 103 (50%) of the culture-positive samples. Compared with culture results, Lubeck's 16S PCR assay had the best performance characteristics, with a sensitivity of 82% and a specificity of 94%. Of the 12 culture-negative samples positive by Lubeck's PCR assay, 11 (92%) samples were also positive by Denis' 16S PCR assay, suggesting that in these cases the culture might have been falsely negative. Based on our results, we conclude that the optimal conditions for detecting Campylobacter spp. in natural waters include 2000 mL sample volume and a single enrichment broth of 25 mL PB incubated for 48 h.     

Selective enrichment media bias the types of Salmonella enterica strains isolated from mixed strain cultures and complex enrichment broths

For foodborne outbreak investigations it can be difficult to isolate the relevant strain from food and/or environmental sources. If the sample is contaminated by more than one strain of the pathogen the relevant strain might be missed. In this study mixed cultures of Salmonella enterica were grown in one set of standard enrichment media to see if culture bias patterns emerged. Nineteen strains representing four serogroups and ten serotypes were compared in four-strain mixtures in Salmonella-only and in cattle fecal culture enrichment backgrounds using Salmonella enrichment media. One or more strain(s) emerged as dominant in each mixture. No serotype was most fit, but strains of serogroups C2 and E were more likely to dominate enrichment culture mixtures than strains of serogroups B or C1. Different versions of Rappaport-Vassiliadis (RV) medium gave different patterns of strain dominance in both Salmonella-only and fecal enrichment culture backgrounds. The fittest strains belonged to serogroups C1, C2, and E, and included strains of S. Infantis, S. Thompson S. Newport, S. 6,8:d:-, and S. Give. Strains of serogroup B, which included serotypes often seen in outbreaks such as S. Typhimurium, S. Saintpaul, and S. Schwarzengrund were less likely to emerge as dominant strains in the mixtures when using standard RV as part of the enrichment. Using a more nutrient-rich version of RV as part of the protocol led to a different pattern of strains emerging, however some were still present in very low numbers in the resulting population. These results indicate that outbreak investigations of food and/or other environmental samples should include multiple enrichment protocols to ensure isolation of target strains of Salmonella.     

Evaluation of two automated enzyme-immunoassays for detection of thermophilic campylobacters in faecal samples from cattle and swine.

We evaluated the performance of two enzyme-immunoassays (EIA) for the detection of naturally occurring, thermophilic Campylobacter spp. found in faecal samples from cattle (n = 21 and n = 26) and swine (n = 43) relative to the standard culture method, and also assuming that none of the tests was the definitive standard. The primary isolation both for the culture and the EIA methods was carried out by overnight selective enrichment in Preston broth. The results showed good sensitivities for both EIA methods in cattle (95% and 84%) and swine (88% and 69%) samples. However, when testing cattle samples, EIA-2 method resulted in a rather low specificity (32%). This seemed to be partially due to the isolation of nonthermophilic species. In conclusion, EIA-1 method may provide a simple and fast tool with good accuracy in cattle and swine samples for automated screening of large number of samples.     

Laboratory study of several enrichment broths for the detection of Salmonella spp. particularly in relation to water samples

The selectivity and efficiency of several enrichment broths used for the detection of salmonellas were comparatively evaluated under laboratory and environmental conditions. Media with selenite were less efficient in their inhibition of the growth of Gram-positive micro-organisms. Salmonellas grew slowly in tetrathionate broth and in media containing brilliant green. These media inhibited the growth of Salmonella typhi, which grew only in media containing selenite. The results obtained in the experiments with stressed salmonellas indicate that the media selenite F, selenite F with novobiocin, selenite cystine and Rappaport-Vassiliadis (RV/43), in conjunction with the double agar layer technique, showed an optimal efficiency for the detection of stressed salmonellas. When natural samples (freshwater and seawater) were used to evaluate the media, however, those containing malachite green, whether or not supplemented with sodium novobiocin, enhanced the recovery of salmonellas.     

Comparative study of membrane filtration and enrichment media for the isolation and enumeration of Pseudomonas aeruginosa from sewage, surface water, and swimming pools

The recovery of Pseudomonas aeruginosa on several selective culture media was tested using raw sewage and secondary sewage effluent samples as well as spiked chlorinated imitation swimming water and samples from whirlpools. mPA-medium B gave good recovery of both vital and chlorine-injured P. aeruginosa and selectivity was greater than 90% when analysing whirlpool samples. It is therefore the medium recommended for examination of chlorinated swimming pools. When analysing sewage polluted water with the mPA-B medium, reduced selectivity was noted from low verification rates and from overgrowth by competitive flora. A modified medium (mPA-D; addition of cetrimide, omission of sulphapyridine and actidione) was more selective and sufficiently recovered noninjured cells. Chlorine-injured cells were completely inhibited, however. C-390 (9-chloro-9-(4-diethylaminophenyl)-10-phenylacridan) was confirmed to be highly selective for P. aeruginosa when used in spread plates at a concentration of 30 micrograms/mL; P. aeruginosa was slightly inhibited. However, the medium could not be used with conventional membrane filtration techniques, because cellulose ester filters interfered with the selective action of C-390. Selectivity could be improved by using Gelman Tuffryn (polysulphone) filters and increasing the C-390 concentration to 120 micrograms/mL. At this concentration, however, the medium was strongly inhibitory to P. aeruginosa; resuscitation only partially improved recovery. Two other membrane filtration media were tested. Both cetrimide - nalidixic acid agar and Drake's medium No. 19 were inhibitory to chlorine-injured cells. Several types of membrane filters were tested and there was little difference between them.(ABSTRACT TRUNCATED AT 250 WORDS)     

Procedure for isolation of Thienamycin from fermentation broths

An improved process for the isolation of thienamycin, produced by the actinomycete Streptomyces cattleya, has been developed. The isolation procedure consists of three chromatographic steps, volume reduction by reverse osmosis between the steps, and freezedrying for obtaining the final product. The chromatographic steps are as follows: (1) ion exchange chromatography on Dowex 1 × 2 resin in the bicarbonate cycle, (2) gel chromatography on Dowex 1 × 2 resin in the chloride cycle, (3) reverse phase chromatography on XAD-2 resin. This procedure is useful for processing large volumes of fermentation broth.     

Towards an international standard for PCR-based detection of foodborne thermotolerant campylobacters: interaction of enrichment media and pre-PCR treatment on carcass rinse samples

As part of a large EU project for standardisation of polymerase chain reaction (PCR), a systematic evaluation of the interaction of enrichment media, type of DNA polymerase and pre-PCR sample treatment for a PCR detecting thermotolerant campylobacters was carried out. The growth-supporting capacity and PCR compatibility of enrichment in Preston, Mueller-Hinton and Bolton broth (blood-containing and blood-free) were evaluated. The effect of resin-based DNA extraction and DNA extraction by boiling on the final PCR assay was investigated. The time-course studies indicated that a 20-h sample enrichment in blood-containing Bolton broth, followed by a simple resin-based extraction of DNA and a PCR amplification using Tth polymerase, resulted in strong and clear PCR amplicons for target (287 bp) and internal amplification control (IAC, 124 bp). The enrichment PCR-based method, tested on 68 presumably naturally contaminated poultry-rinse samples, showed a diagnostic sensitivity of 97.5% (39 PCR-positive/40 total positive samples) and a diagnostic specificity of 100% (28 PCR-negative/28 total negative samples; P=0.32) when compared to a standard bacteriological method (ISO 10272).     

Suitability of some enrichment broths and diluents for enumerating cold- and heat-stressed Vibrio parahaemolyticus.

Vibrio parahaemolyticus cells were injured by chilling and heating, and their recovery was tested in glucose-salt-Teepol broth (GSTB), tryptic soy broth containing 7% NaCl (TSBS), Horie - arabinose - ethyl violet broth (HAEB), and water blue - alizarin yellow broth (WBAY). Exponential phase cells were more sensitive to cold shock than were stationary phase cells. Exposure of chill-injured V. parahaemolyticus to GSTB and TSBS resulted in 70 to 80% death; about 70% lethality was noted for heat-injured cells inoculated into TSBS. Neither HAEB nor WBAY enrichment media were lethal to stressed cells, although rates of growth were retarded. The 3% NaCl in 0.1 M potassium phosphate (pH 7.0) diluent proved to be most suitable for protecting against inactivation of cold- and heat-injured cells.