Comparison of three rapid screening methods for Salmonella spp.: 'MUCAP Test, MicroScreenR Latex and Rambach Agar'

Three new rapid methods for detection of Salmonella spp. have been studied. The fluorogenic MUCAP test (Biolife, Italy), the MicroScreen^R latex slide agglutination test (Mercia, UK) and the Rambach agar test (Technogram, France) were compared for their sensitivity and their specificity. Some 175 strains, incuding 74 Salmonella strains and 101 non- Salmonella strains were included in the study. The sensitivities of the MUCAP, the MicroScreen^R and Rambach agar tests were 100%, 96% and 91%, respectively, and their specificities 80%, 96% and 100%, respectively.     

Note: Comparison of media for the isolation of lactose-positive Salmonella

We studied the capacity of 10 selective media (Rambach agar, RB; salmonella-shigella agar, SS; SM-ID medium, SM; Hektoen enteric agar, HE; modified semisolid Rappaport-Vassiliadis agar, MSRV; bismuth sulphite agar, BS; MacConkey agar, MC; brilliant green agar, BG; novobiocin-brilliant green-glucose agar, NBG; and novobiocin-brilliant green-glycerol-lactose agar, NBGL), and the C₈-esterase test (MUCAP test, Biolife, Italy) to detect the growth of 14 strains of lactose-positive Salmonella (12 Salm. virchow and two Salm. montevideo ) and 16 Salm. arizonae. Suspensions of pure strain were plated on the aforementioned media and on Mueller-Hinton, used as a control, with inocula of 3 x 10² cfu ml^-1. The performance of BS was excellent, determining the 30 strains as typical Salmonella colonies (H₂S+). On NBG, 27 strains were detected. On MSRV, only some strains grew and only one produced swarming. On the other media, the two Salm. montevideo and the 12 Salm. virchow strains produced coliform colonies. Some of these latter were inhibited on BG and NBGL. The 16 Salm. arizonae strains produced typical colonies on all the media, except on RB, SM and MSRV. On NBGL, two strains did not produce H₂S. The C₈-esterase test was only successful with Salm. montevideo and Salm. virchow on NBG and RB (with a few exceptions on the latter). However, with Salm. arizonae the test was positive on SS, MC, HE, BG and NBG. In summary, BS was the best medium of those used (all the 30 strains were isolated), followed by NBG (27 isolates).     

Evaluation of Rambach agar for the differentiation of Salmonella species from other Enterobacteriaceae

Rambach agar (Merck) was evaluated for its reliability as a selective diagnostic medium for the differentiation of Salmonella species from other Enterobacteriaceae. Twenty-five Salmonella strains were cultured on each of three agar media, Rambach (RAM), xylose lysine desoxycholate (XLD) and bismuth sulphite (BSA). Typical, easily interpreted reactions and colony morphologies were achieved for 23 strains on RAM and BSA and 17 on XLD. Of 135 other Enterobacteriaceae cultured on RAM, 134 gave characteristics which differentiated them from Salmonella . One strain which looked like Salmonella was identified as Citrobacter freundii . Rambach agar has potential as a supplementary agar in testing foods for Salmonella , but as with other selective diagnostic agars, it has limitations.     

应用MUCAP试剂快速检测沙门氏菌

报告了用4-甲基伞形酮辛酯(4-Methylumbelliferyl-caprylate, MUCAP)快速检测沙门氏菌的特异性、敏感性和实用性。经HE,DHL,SS和麦康凯琼脂平板分离的65株沙门氏菌标准菌株和48株从食品中分离的沙门氏菌,用MUCAP测试均呈阳性反应;394株非沙门氏菌中呈阳性反应的假单胞菌、气单胞菌、邻单胞菌可通过氧化酶试验与沙门氏菌区分开;与粘质沙雷氏菌的交叉反应改用加1％蔗糖的分离平板也可排除。此方法的敏感性和特异性均达到97％以上,而且操作简便、快速,数分钟内即可完成。     

Evaluation of new culture media for rapid detection and isolation of salmonellae in foods.

Conventional methods for Salmonella detection in foods can require up to 6 and at least 4 days. We have observed that the total analysis time can be reduced to 48 h by using Salmosyst broth as a liquid medium for both preenrichment and selective enrichment and Rambach agar (RA), a new selective plate medium. In samples of artificially contaminated ground beef Salmonella enteritidis was detected at a concentration of 0.4 CFU/g (10 CFU/25 g) by both a conventional method and the new method. Of 519 samples of foods for sale, 38 were Salmonella positive by both methods while 471 were negative. Nine samples which were negative by the conventional method were positive by the Salmosyst-RA method, while one sample positive by the first method was negative by the last. Therefore, the Salmosyst-RA method showed 97.9% sensitivity compared with the 81.2% sensitivity of the conventional method. The new method was also highly specific (98% specificity) in presumptive identification of Salmonella colonies. Furthermore, a 6-h preenrichment in Salmosyst broth has been proved sufficient for the repair of heat-injured Salmonella cells and for subsequent recovery by selective enrichment. In conclusion, the Salmosyst-RA method shows several advantages over both conventional and rapid noncultural methods: (i) only two media are required instead of the five media for conventional methods; (ii) in real time it is comparable to other rapid noncultural methods, which require 30 to 31 h; (iii) it is highly sensitive and specific; and (iv) it allows the isolation of Salmonella strains which can be characterized by appropriate phenotypic and genotypic typing methods for epidemiological investigations.     

Detection of Salmonella by indicator agar media and PCR as affected by alfalfa seed homogenates and native bacteria

AIMS: To investigate and prevent the undesirable effect of native bacteria and alfalfa seed homogenates on detection of Salmonella in alfalfa seeds by indicator agar media and polymerase chain reaction (PCR). METHODS AND RESULTS: The relative sensitivity of five indicator agar media, including modified semisolid RV (MSRV), xylose-lysine-Tergitol 4 (XLT4), Hektoen enteric agar (HEA), brilliant green agar (BGA) and bismuth sulphite agar (BSA), for detection of Salmonella in the presence of a large number of native bacteria from alfalfa seeds was examined. The detection limit as measured by the ratio between the numbers of native bacteria and Salmonella was estimated to be 10(6) to 1 for MSRV and 10(3) to 1 for XLT4, HEA, BGA or BSA. Presence of alfalfa seed homogenates markedly reduced the sensitivity of Salmonella detection by PCR. The minimal number of Salmonella detectable by PCR was determined to be 1-10 and 100-1000 CFU in the absence and presence of seed homogenate, respectively. Application of anti-Salmonella immunomagnetic beads permitted detection of 2-5 CFU of heat-injured cells in 25 g of seeds within 24 h by PCR. CONCLUSIONS: The MSRV medium is more sensitive than other indicator agars for detecting a small number of motile Salmonella in samples containing a large number of native bacteria. Application of immunomagnetic beads eliminates the PCR-inhibitory activity of seed homogenates and improves the detection of Salmonella in inoculated seeds. SIGNIFICANCE AND IMPACT: The results generated from this study will aid the seed distributors, sprout growers and public health officials to identify and recall the Salmonella-contaminated seed lots to be used for sprout production.     

Isolation of Shigellae: VIII. Comparison of Xylose Lysine Deoxycholate Agar, Hektoen Enteric Agar, Salmonella-Shigella Agar, and Eosin Methylene Blue Agar with Stool Specimens

Two enrichment broths and four plating media were compared for efficiency of detection of enteric pathogens from 1,597 stool specimens. Of 170 salmonellae isolated from the composite of all methods, direct streaking yielded but 54%, whereas enrichment in gram-negative broth found 87% and Selenite-F broth 97%. By contrast, gram-negative broth produced 100% of the 17 shigellae, Selenite-F broth but 77%, and direct streaking only 59%. Thus, enrichment methods produced almost twice the number of both pathogens as direct streaking. Comparison of the plating media revealed xylose lysine deoxycholate agar (XLD) and Hektoen enteric agar to be equal in their abilities to find both pathogens. Both were moderately better than Salmonella-Shigella agar and markedly superior to eosin methylene blue agar. XLD fround 83% of salmonellae produced by the composite of four media and 90% of the shigellae. Hektoen enteric agar found 80% of both. Salmonella-Shigella agar detected 74 and 68%, respectively, and eosin methylene blue agar only 42 and 63%. The numbers of false positives accruing to each medium, however, showed Hektoen enteric and Salmonella-Shigella agars to produce more than twice as many false-positive plates as XLD. Similarly, Selenite-F broth resulted in many more false-positives for all plating media than did gram-negative broth. Consequently, the index of validity, which equates successful isolation of pathogens with total pickings, favored XLD and gram-negative broth as the media of choice, with direct streaking the poorest method by all counts.     

Evaluation of Rambach agar for detection of Salmonella subspecies I to VI.

Salmonella strains belonging to subspecies I to VI were investigated for colony color when grown on Rambach agar. Most strains of Salmonella subspecies I, II, IV, and VI behaved as described. All strains of Salmonella subspecies IIIa, IIIb, and V produced beta-D-galactosidase and blue-green colonies which could not be distinguished in color from Escherichia coli and other lactose-fermenting members of the family Enterobacteriaceae.     

Assessment of the presence of Salmonella spp. in Egyptian dairy products using various detection media

AIMS: To make a preliminary assessment of the incidence of Salmonella in Egyptian dairy products, and to investigate the effectiveness of various protocols for the detection of the pathogen in these products. METHODS AND RESULTS: Samples of milk and related dairy products were randomly collected from local markets and examined for the presence of Salmonella. While most samples were free of the organism, isolates of Salmonella enterica subsp. enterica serovar Typhimurium PT 8 could be recovered from 'matared' cream specimens. These isolates were susceptible to antibiotics usually used to challenge infections caused by Salmonella. A combination of buffered peptone water, Muller-Kauffman tetrathionate broth, and brilliant green phenol red agar gave the best results for the detection of the pathogen. Selenite-cystine broth and Hektoen enteric agar were ineffective as an enrichment and a plating medium, respectively, in the isolation of Salmonella. A modified identification strategy that reduces the burden of serological testing of presumptive isolates is proposed. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: 'Matared' cream could be a vehicle for transmitting Salmonella. Using the above combination of media, beside the suggested modified confirmatory procedure, should increase the effectiveness and ease of the detection of Salmonella in milk and dairy products.     

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.     

Comparative study on Salmonella isolation from sewage—contaminated natural waters

A lcaide , E., M artinez , J.P. & G aray , E. 1984. Comparative study on Salmonella isolation from sewage contaminated natural waters. Journal of Applied Bacteriology 56 , 365–371.
A comparative study of five factors influencing the isolation of salmonellas from sewage-contaminated natural waters was carried out. The effect of pre-enrichment in buffered peptone water was compared with single-step enrichment in NR10 broth incubated at 43C. A modification of NR10 has been compared with the original composition. Bismuth sulphite agar (BSA), Hektoen enteric agar (HE) and brilliant green agar (BGA) have been used as plating media. Other factors considered have been temperature of the water and sampling site. A total of 759 salmonella strains belonging to 36 different serotypes has been recovered in a two-year study. All five factors considered in the study have shown a significant effect on the recovery of salmonellas. The combination of direct enrichment in NR10, followed by BSA or HE as plating media was most effective for the isolation of Salmonella . The influence of water temperature and characteristics of the sampling sites have also been discussed.     

Comparison of the MSRV method with an in-house conventional method for the detection of Salmonella in various high and low moisture foods

In this trial an in-house conventional method was compared with the MSRV method for Salmonella detection. Various high and low moisture foodstuffs (121 and 116 samples respectively), some either artificially or naturally contaminated, were examined.
Eleven different serotypes of Salmonella were used to inoculate samples and no significant difference was observed in the sensitivity of any of the media used. Significantly lower numbers of false-positive results were obtained with the MSRV agar when compared to MLCB agar.
This work suggests that the MSRV method as used here, could be used to replace conventional Salmonella detection methods for both high and low moisture foodstuffs.     

Observations on brilliant green agar with H2S indicator.

Several formulations of brilliant green agar with an added H2S indicator were evaluated. Results were optimum with variations of a basic formula consisting of 40 g of tryptic soy agar (Difco), 8 g of lactose, 8 g of sucrose, 80 mg of phenol red, 1 g of sulfanilamide, 1.5 g of ferric ammonium citrate, 5 g of sodium thiosulfate pentahydrate, and 7 mg of brilliant green dye per liter. Brilliant green dye was added after sterilization of the other components This formulation supported good growth of all of 39 strains of Salmonella tested. Normal biochemical types formed pink colonies with black centers, and an H2S-negative S. choleraesuis formed pink colonies without black centers. Of other bacteria tested, only Enterobacter, Klebsiella, and a few Citrobacter strains showed significant growth in 24 h. When lactose was omitted from the formulation, a lactose-fermenting strain formed pink colonies with black centers, and differentiation of Salmonella from the Enterobacter-Klebsiella groups was equally good. Addition of xylose (4.0 g) and L-lysine hydrochloride (5.4 g) to the above formulation improved differentiation between Salmonella and the few Citrobacter strains that grew and produced more intense blackening in Salmonella colonies. Addition of an H2S indicator to brilliant green agar formulations aided in identification of Salmonella colonies, especially in mixtures with other bacteria. These media were judged to give better differentiation of salmonellae from other bacteria than Hektoen agar with added novobiocin (10 mg/liter).     

Observations on brilliant green agar with H2S indicator.

Several formulations of brilliant green agar with an added H2S indicator were evaluated. Results were optimum with variations of a basic formula consisting of 40 g of tryptic soy agar (Difco), 8 g of lactose, 8 g of sucrose, 80 mg of phenol red, 1 g of sulfanilamide, 1.5 g of ferric ammonium citrate, 5 g of sodium thiosulfate pentahydrate, and 7 mg of brilliant green dye per liter. Brilliant green dye was added after sterilization of the other components This formulation supported good growth of all of 39 strains of Salmonella tested. Normal biochemical types formed pink colonies with black centers, and an H2S-negative S. choleraesuis formed pink colonies without black centers. Of other bacteria tested, only Enterobacter, Klebsiella, and a few Citrobacter strains showed significant growth in 24 h. When lactose was omitted from the formulation, a lactose-fermenting strain formed pink colonies with black centers, and differentiation of Salmonella from the Enterobacter-Klebsiella groups was equally good. Addition of xylose (4.0 g) and L-lysine hydrochloride (5.4 g) to the above formulation improved differentiation between Salmonella and the few Citrobacter strains that grew and produced more intense blackening in Salmonella colonies. Addition of an H2S indicator to brilliant green agar formulations aided in identification of Salmonella colonies, especially in mixtures with other bacteria. These media were judged to give better differentiation of salmonellae from other bacteria than Hektoen agar with added novobiocin (10 mg/liter).     

Detection of Salmonella strains and Escherichia coli O157:H7 in feces of small ruminants and their isolation with various media

Salmonella strains and Escherichia coli O157:H7 were detected in 17 and 5 small ruminants in Virginia, respectively, of 287 tested. Background microflora interfered with the fecal analysis. The combination of Salmonella enzyme immunoassay (EIA) detection and xylose-lysine-deoxycholate agar isolation was satisfactory. Modifying enrichment to a 1:100 dilution enabled effective E. coli O157:H7 detection by EIA and isolation by sorbitol-MacConkey agar with cefixime-tellurite.     

Inhibitory Action of Various Plating Media on the Growth of Certain Salmonella Serotypes

The growth of 68 strains of Salmonella typhi , 697 other Salmonella strains and 213 strains of other Gram negative intestinal bacteria on 8 plating media was assessed semi-quantitatively. These media were found to be differentially inhibitory to different Salmonella serotypes. The combined use of two plating media, brilliant green MacConkey agar and deoxycholate citrate agar, allowed potentially the recovery of the maximum number of Salmonella strains. If only one medium was used, brilliant green MacConkey agar would appear to be the best plating medium for the isolation of non-typhoid salmonellas in general and S. choleraesuis in particular. Xylose lysine deoxycholate agar, on which a certain proportion of salmonellas failed to yield typical, recognizable colonies, was found not to be a good plating medium for their isolation.     

Fluorogenic assays for immediate confirmation of Escherichia coli.

Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).     

Quantitative evaluation of growth-promoting properties of selected culture media used for isolation of Salmonella and Shigella strains

Growth promoting properties and selectivity of 11 commercially produced media recommended for Salmonella and Shigella isolation were evaluated. The following media were tested: EMB (Eosin methylene blue agar), Endo, P?oskiriew, MacConkey, DC (Deoxycholate citrate agar), SS (Salmonella-Shigella agar), BS (Bismuth sulfite agar) and Mueller-Hinton as a medium with no selective properties. The media were produced in Czechoslovakia, East Germany, West Germany, Poland, and Soviet Union. Quantitative studies were performed on 71 strains representing 8 genera of Enterobacteriaceae family; both reference and wild newly + isolated from clinical material strains were included. It was found that none of DC and BS media provided suitable growth conditions for Shigella strains and in particular for S. dysenteriae, S. boydii, and S. flexneri. It was also found that the same medium (name and content) but derived from different producer can vary significantly in respect to growth promotion and selectivity especially for Shigella strains. All media with selective, differentiating properties for Salmonella and Shigella isolation should not be used without previous quantitative control test for their selective and growth promoting properties checked by user. The need for such a control performed both on reference and freshly isolated strains was shown in this study. In the set of control strains all species of Shigella should be represented.     

Some Observations on the Incorporation of Novobiocin into Hektoen Enteric Agar for Improved Salmonella Isolation

Novobiocin was incorporated into Hektoen enteric agar as a means of suppressing those Proteus and Citrobacter organisms that produce colonies which are often mistaken for Salmonella.     

Evaluation of different plating media used in the isolation of salmonellas from environmental samples

Different serotypes of salmonellas were compared for selectivity and efficiency of recovery using 11 plating media. No optimal growth was obtained after 24 h incubation in any of the media, but after 48 h, brilliant green, brilliant green-phenol red-lactose-sucrose, bismuth sulphite, xylose-lysine-deoxycholate and Hektoen enteric agars showed optimal recovery of all the salmonella serotypes. Xylose-lysine-deoxycholate and brilliant green-phenol red-lactose-sucrose agars were the most selective media for all salmonella serotypes. Addition of 10 micrograms/ml of sodium novobiocin to the tryptic soy-xylose-lysine and tryptic soy-brilliant green agars significantly improved their selectivity but reduced or inhibited the growth of some salmonella serotypes, including Salmonella typhi. Xylose-lysine-deoxycholate agar gave the highest recovery percentage of stressed salmonellas with a double-agar layer technique. Good recovery was also obtained on brilliant green-phenol red-lactose-sucrose, tryptic soy-brilliant green, tryptic soy-brilliant green-novobiocin, tryptic, soy-xylose-lysine and tryptic soy-xylose-lysine-novobiocin agars. Salmonella-shigella agar was the least efficient medium for the recovery of salmonellas under stress-induced or non-stressed conditions.