Application of the rapid lysine decarboxylase test for early isolation and detection of salmonellae in sewage and other wastewaters.

A method for early isolation and detection of salmonellae in sewage and other wastewaters by using the rapid lysine decarboxylase test as a single biochemical reaction for screening the suspected Salmonella colonies is described. By this method, Salmonella isolation and identification can be completed within 2 to 3 days in contrast to the 5 to 7 days required for the conventional method.     

Rapid radiometric method for detection of Salmonella in foods.

A radiometric method for the detection of Salmonella in foods has been developed which is based on Salmonella poly H agglutinating serum preventing Salmonella from producing 14CO2 from [14C]dulcitol. The method will detect the presence or absence of Salmonella in a product within 30 h compared to 4 to 5 days by routine culture methods. The method has been evaluated against a routine culture method using 58 samples of food. The overall agreement was 91%. Five samples negative for Salmonella by the routine method were positive by the radiometric method. These may have been false positives. However, the routine method may have failed to detect Salmonella due to the presence of large numbers of lactose-fermenting bacteria which hindered isolation of Salmonella colonies on the selective agar plates.     

Rapid detection of Salmonella spp. in food by use of the ISO-GRID hydrophobic grid membrane filter.

A rapid hydrophobic grid-membrane filter (HGMF) method was developed and compared with the Health Protection Branch cultural method for the detection of Salmonella spp. in 798 spiked samples and 265 naturally contaminated samples of food. With the HGMF method, Salmonella spp. were isolated from 618 of the spiked samples and 190 of the naturally contaminated samples. The conventional method recovered Salmonella spp. from 622 spiked samples and 204 unspiked samples. The isolation rates from Salmonella-positive samples for the two methods were not significantly different (94.6% overall for the HGMF method and 96.7% for the conventional approach), but the HGMF results were available in only 2 to 3 days after sample receipt compared with 3 to 4 days by the conventional method.     

Development of liposome immunoassay for salmonella spp. using immunomagnetic separation and immunoliposome

The ability to detect Salmonella spp. is essential in the prevention of foodborne illness. This study examined a Salmonella spp. detection method involving the application of immunomagnetic separation and immunoliposomes (IMS/IL) encapsulating sulforhodamine B (SRB), a fluorescent dye. A quantitative assay was conducted by measuring the fluorescence intensity of SRB that was produced from an immunomagnetic bead-Salmonella spp.-immunoliposome complex. The results indicated detection limits of 2.7x10(5) and 5.2x10(3) CFU/ml for Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) and Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium), respectivley. The signal/noise ratio was improved by using 4% skim milk as a wash solution rather than 2% BSA. In addition, higher fluorescence intensity was obtained by increasing the liposome size. Compared with the conventional plating method, which takes 3-4 days for the isolation and identification of Salmonella spp., the total assay time of 10 h only including 6 h of culture enrichment was necessary for the Salmonella detection by IMS/IL. These results indicate that the IMS/ IL has great potential as an alternative rapid method for Salmonella detection.     

Isolation of Salmonella strains from reptile faeces and comparison of different culture media

AIMS: To provide information on epidemiology and isolation of Salmonella strains from reptiles. METHODS AND RESULTS: Ninety-one samples collected from reptiles of the zoo of Rome or belonging to private owners were analysed using a standard protocol for isolation of Salmonella from food. Salmonella strains were tested for susceptibility to 15 antimicrobics by a disc-agar diffusion method. Forty-six samples (50.5%) were positive for Salmonella. Of the 22 strains serotyped, 17 belonged to Salmonella enterica subsp. I, four to the subsp. IIIa and one strain resulted untypeable. Rappaport-Vassiliadis broth (RVB) allowed to recover more Salmonella strains when bacterial growth in buffered peptone water (BPW) was scarce, while selenite cystine broth (SCB) was more efficient, whereas growth in BPW was abundant. The maximum isolation score was obtained by plating onto xylose lysine desoxycholate agar (XLD). The strains exhibited resistance at high percentages to colistin sulphate (58.7%), sulphamethoxazole (55.5%), streptomycin (32.6%), tetracycline (19.6%), ampicillin (17.4%) and nalidixic acid (13.1%). CONCLUSIONS: A high prevalence of Salmonella in reptiles was observed. For isolation, the choice of the enrichment broth depending on the degree of growth in BPW followed by plating onto XLD may be suggested. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides epidemiological data on the prevalence of Salmonella and laboratory protocols useful for isolation of Salmonella from faeces of reptiles.     

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.     

2010 年上海市食源性沙门菌菌型分布和药敏分析 以及快速检测方法的建立

目的:通过对2010年上海市市售食品中食源性沙门菌污染状况、菌型分布及药敏试验进行分析,初步确定该地区食源性沙门菌的菌型分布及耐药性,为防治因沙门菌感染引起的食源性疾病提供科学依据,并摸索建立了针对沙门菌的快速检测方法。方法:对农贸市场和超市的5类840份市售食品进行沙门菌分离鉴定及耐药性分析,并根据沙门菌的侵袭蛋白A(invA)基因序列设计保守引物,特异性快速检测沙门菌。结果:本次市售食品沙门菌阳性率检出率为4.29%,共36株沙门菌,生禽畜肉所占比例高达91%。主要血型为鼠伤寒沙门菌,德尔卑沙门菌和都柏林沙门菌。36株沙门菌药敏分析显示:所有菌株对头孢类等抗生素敏感较高,但对氨苄西林、麦迪霉素、环丙沙辛、呋喃妥因等存在普遍较多耐药菌株,并在此基础上通过PCR法成功地特异性检测出沙门菌。结论:上海市市售食品沙门菌污染以生禽畜肉为主,对抗菌药物的耐药性较高。目前治疗市售食品中食源性沙门菌引起的食源性疾病应首选头孢内等抗生素。另外PCR快速检测方法也操作简单,特异性强,灵敏度高,对食品中沙门菌污染能起到快速检测和监控。     

Rapid procedure for isolation and detection of Salmonella plasmids

A rapid procedure is described for the isolation of high quality and quantity plasmid DNA from Salmonella. Plasmids of molecular mass ranging from 10 kbp to 147 kbp were isolated. The method yielded enough DNA from the low copy number virulence plasmids of various Salmonella serovars for visualization on an agarose gel. The method is based on the classic alkaline lysis methods, employing the same reagents, but without any incubation steps. Isolation of plasmid DNA was also attempted with the use of rapid DNA isolation kits, but without success. It was reasoned that endonucleases, present in the resin, might have degraded the plasmids. © Rapid Science Ltd. 1998     

Comparison of commercially available kits with standard methods for detection of Salmonella strains in foods.

Six commercial kits were compared with the U.S. Food and Drug Administration (USFDA) method and the Japanese standard method for Salmonella isolation in foods. When only Salmonella serovars were tested, many of the methods performed well; however, when foods were artificially inoculated, only the USFDA method and immunomagnetic separation coupled with the xylose-lysine-brilliant green agar method (MS-XLBG) could positively detect Salmonella serovars. All seven wild-type Salmonella serovars were detected by the USFDA method, and the MS-XLBG method detected salmonellae from six samples.     

Isolation of Salmonella from environmental samples collected in the reptile department of Antwerp Zoo using different selective methods

AIMS: To evaluate the environmental spread of Salmonella strains in the reptile department of Antwerp Zoo and to compare different isolation methods for Salmonella. METHODS AND RESULTS: One hundred environmental samples were collected in the service sections and public spaces of the reptile department. After pre-enrichment in buffered peptone water (BPW), selective enrichment was performed in Rappaport Vassiliadis Single Component Enrichment Broth (RVS), Selenite Cystine Broth (SEL) and Mueller Kauffman Tetrathionate Broth (MKTTn). Subculturing on Modified Semisolid Rappaport-Vassiliadis (MSRV) Medium, and the combined use of immunomagnetic separation (IMS) and RVS was evaluated. The isolation media used were Hektoen Enteric Agar (HE), Phenol Red Brilliant Green Agar (BG) and Xylose Lysine Decarboxylase Agar (XLD). Salmonella strains were found in 47 samples (47.0%). Most isolations were made on HE after combined IMS/RVS enrichment. Sixty-six Salmonella strains were serotyped, 29 belonged to Salmonella enterica ssp. enterica (I), 3 to ssp. salamae (II), 29 to ssp. arizonae or diarizonae (IIIa/b), 4 to ssp. houtenae (IV) and 1 strain showed autoagglutination. In addition, a 10-year survey (1995-2004) of Salmonella serovars isolated from reptiles at Antwerp Zoo is presented. CONCLUSIONS: A high prevalence of Salmonella strains was noted in the service sections of the reptile department. Only a few isolations were made in the public spaces. Selective enrichment in RVS was the most efficient. In combination with IMS, this method gave an even higher isolation rate than the International Standard method (ISO 6579:2002). SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms the importance of reptiles as spreaders of Salmonella in their surroundings. The possible infectious risks for zoo personnel and visitors are evaluated. Improved laboratory protocols for the isolation of Salmonella from the environment are suggested.     

Evaluation of Methods for Isolating Salmonella and Arizona Organisms from Pet Turtles

Two methods for isolating Salmonella and Arizona organisms from turtles, blending and excretion, were evaluated, and the percentage of isolates obtained by each method was compared with the percentage of isolates obtained by culture of turtle organs. The blending and excretion methods were equally effective in detecting the overall incidence of Salmonella and Arizona infections in turtles. The percentage of isolates obtained by specific organ culture, however, was less than the percentage obtained by the other two methods. The blending method detected a greater number of turtles with Arizona infections than did the excretion method, but there was no difference in the number of Salmonella infections detected by the two methods. The frequency of isolation of Arizona organisms from organs other than the small intestine and colon was higher than that of Salmonella.     

2010年夏季急性细菌感染性腹泻患儿沙门菌感染分析

本文旨在了解急性细菌感染性腹泻患儿中沙门菌的感染现状及其耐药性.采集2010年7月1日~2010年9月30日复旦大学附属儿科医院门诊及住院急性细菌感染性腹泻患儿的粪便标本,进行沙门菌分离、培养及鉴定,采用Kirby-Bauer法进行药敏试验.共收集急性细菌感染性腹泻患儿粪便标本 1 045份,检出沙门菌 160株,阳性...     

2010年上海市食源性沙门菌菌型分布和药敏分析以及快速检测方法的建立

目的：通过对2010年上海市市售食品中食源性沙门菌污染状况、菌型分布及药敏试验进行分析,初步确定该地区食源性沙门菌的菌型分布及耐药性,为防治因沙门菌感染引起的食源性疾病提供科学依据,并摸索建立了针对沙门菌的快速检测方法。方法：对农贸市场和超市的5类840份市售食品进行沙门菌分离鉴定及耐药性分析,并根据沙门菌的侵袭蛋白A（invA）基因序列设计保守引物,特异性快速检测沙门菌。结果：本次市售食品沙门菌阳性率检出率为4．29％,共36株沙门菌,生禽畜肉所占比例高达91％。主要血型为鼠伤寒沙门菌,德尔卑沙门菌和都柏林沙门菌。36株沙门菌药敏分析显示：所有菌株对头孢类等抗生紊敏感较高,但对氨苄西林、麦迪霉素、环丙沙辛、呋喃妥因等存在普遍较多耐药菌株,并在此基础上通过PCR法成功地特异性检测出沙门菌。结论：上海市市售食品沙门菌污染以生禽畜肉为主,对抗菌药物的耐药性较高。目前治疗市售食品中食源性沙门菌引起的食源性疾病应首选头孢内等抗生素。另外PCR快速检测方法也操作简单,特异性强,灵敏度高,对食品中沙门菌污染能起到快速检测和监控     

Retrospective and longitudinal study of salmonellosis in captive wildlife in Trinidad

Morbidity and mortality of captive wildlife at the Emperor Valley Zoo, Trinidad from 1993 to 1996 were analysed to determine involvement of Salmonella spp. A 6 mo longitudinal study was conducted to determine the frequency of isolation of Salmonella spp. from apparently healthy, sick and dead wild mammals, birds, and reptiles. The antibiograms of Salmonella isolates were determined using the disc diffusion method. Fecal samples randomly selected from animal enclosures and cloacal swabs of snakes were cultured for Salmonella spp. following enrichment in tetrathionate and selenite cystine broths. For the 1993-96 period, Salmonella spp. was implicated in 17 (12%) of 141 sick or dead animals and the predominant serotype was S. typhimurium. During the 6 mo prospective study in a mean animal population of 1,186, there were 20 (2%) and 14 (1%) animals that were sick and died respectively; Salmonella spp. was implicated in only one mortality. Overall, of 1,012 samples from apparently healthy wildlife cultured, 66 (7%) yielded 24 serotypes of Salmonella. The predominant serotype were S. seigburg (16 isolates), S. gaminara (6 isolates), and S. thompson (6 isolates). None of the samples yielded S. typhimurium. The frequency of isolation of Salmonella spp. in reptiles (14%) was significantly higher than found in either mammals (7%) or birds (3%). Sixty-five (99%) of 66 Salmonella spp. isolates exhibited resistance to one or more of the nine antimicrobial agents tested. Resistance was high to cephalothin (92%), moderate to streptomycin (35%) and tetracycline (29%), but significantly low to gentamicin (2%), chloramphenicol (0%), and sulphamethoxazole/trimethoprim (0%). The prevalence of asymptomatic infections by Salmonella spp. in zoo animals was high and the very high prevalence of antimicrobial resistance could be a problem when treating salmonellosis.     

Evaluation of real-time PCR vs automated ELISA and a conventional culture method using a semi-solid medium for detection of Salmonella

AIMS: Evaluation of iQ-Check PCR Salmonella for Salmonella detection in artificially and naturally contaminated food and environmental field samples. METHODS AND RESULTS: Artificially contaminated samples (poultry meat and ground red meat) subjected to cold- and freeze-stress, and 120 naturally contaminated samples (swabs and meat) were tested for Salmonella using the diagnostic semi-solid Salmonella medium (DIASALM) method, the Vidas assay and the iQ-Check PCR assay after 24 h enrichment in buffered peptone water. CONCLUSIONS: Both the iQ-Check PCR and the Vidas assay provide a rapid and user friendly screening method for detection of Salmonella. False negative samples were obtained for the inoculated samples using both the iQ-Check PCR assay and the Vidas method when Salmonella cells were severely stressed. In total 45 of 120 naturally contaminated field samples showed Salmonella positive using the DIASALM method. The agreement percentage with the DIASALM method was respectively 92% for the iQ-Check PCR and 95% for the Vidas method. SIGNIFICANCE AND IMPACT OF THE STUDY: False-negative samples were obtained for the inoculated samples using both the iQ-Check PCR assay and the Vidas method when Salmonella cells were severely stressed, e.g. freezing at -18 degrees C for 7 days. Of the 120 naturally contaminated field samples 45 showed Salmonella positive using the DIASALM method. The agreement percentage with the DIASALM method was 92% for the iQ-Check PCR and 95% for the Vidas method respectively.     

Incidence of Campylobacter and Salmonella isolated from retail chicken and associated packaging in South Wales

AIMS: To investigate the incidence of Campylobacter and Salmonella contamination associated with supermarket and butchers' shop chicken and related packaging. METHOD AND RESULTS: Three hundred raw samples (whole chicken, chicken breast with skin or chicken pieces) were purchased on a monthly basis for seven months. Packaging associated with the chicken was also sampled to provide isolation data for external and whole packaging. Campylobacter and Salmonella were isolated from 68% and 29% of retail chicken, respectively. Campylobacter was isolated from 3% of external and 34% of whole packaging overall. Salmonella was absent from external packaging but was isolated from 11% of whole packaging. No significant trends in isolation rates of the organisms were obtained during the period of sampling. CONCLUSIONS: The food industry and consumers should be made aware of the potential risk of Campylobacter and Salmonella on both the external and internal surfaces of packaging in addition to chicken itself. SIGNIFICANCE AND IMPACT OF THE STUDY: Chicken and chicken packaging is a potential vehicle for the introduction of pathogens in retail and domestic kitchens and in particular for the cross-contamination of Campylobacter and Salmonella.     

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°C, Selenite Broth with Brilliant Green and Sulphapyridine at 37°C and 43°C, and Rappaport-Vassiliadis Broth (RV 10) at 42°C. The isolation media were Brilliant Green Agar (BGA), Deoxycholate Citrate Agar, Hektoen Enteric Agar (HEA) and Salmonella-Shigella Agar.
Enrichment in RV/42°C followed by isolation on BGA as recommended by ISO standard no. 6579 and enrichment in TTB/37°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.     

Treatment of Salmonella-Arizona-Infected Turtle Eggs with Terramycin and Chloromycetin by the Temperature Differential Egg Dip Method

Attempts to eliminate Salmonella and Arizona infection from newly hatched turtles were made by dipping fresh eggs in cold solutions of Terramycin and Chloromycetin at 1,000, 1,200, 1,500 and 2,000 mug per ml for either 10, 20, or 30 min. Control groups consisted of hatchings produced from nondipped eggs or eggs dipped in chilled water. In two of the four experiments 5 to 10 eggs were blended on days 15, 30, and 45 post antibiotic dip treatment. Twenty-five to 60 hatchlings from each control or experimental dip groups were held in containers and the water was tested (excretion method) for Salmonella and Arizona every 15 or 30 days for 180 to 210 days after hatching. Representative turtles were homogenized (blending method) to determine if systemic infections were present. All specimens tested were enriched in tetrathionate and selenite cystine broth. Nondipped eggs and water-dipped eggs routinely showed Salmonella and Arizona present in egg homogenate and hatchlings emerging from these eggs excreted these pathogens. Terramycin- and Chloromycetin-dipped eggs were uniformly negative for these pathogens, only if fresh eggs were dipped. Bacteriological assay of container water and whole turtle homogenate from hatchlings were negative for Salmonella and Arizona if eggs were dipped in 1,000 mug of Terramycin early in the egg laying season or if eggs were dipped in 1,500 or 2,000 mug of Terramycin per ml late in the egg laying season. The results of temperature-differential egg dip studies suggest that this is a feasible and promising method by which to eradicate Salmonella and Arizona from the turtle.     

FACS-isolation of Salmonella-infected cells with defined bacterial load from mouse spleen

Salmonella can cause a typhoid fever like disease in genetically susceptible mice. In contrast to in vitro cell culture models, most host cells in infected tissues contain only one or two Salmonella, but a small subpopulation of infected host cells contains many Salmonella. It has been proposed that these various subpopulations have differential relevance during infection but to test this, methods for isolating such Salmonella will be required. We developed a method to purify differentially infected host cells by flow cytometry using Salmonella expressing the green fluorescent protein (GFP). Critical parameters for this method were sufficient GFP expression to detect infected cells against strong host cell autofluorescence, and low variation in GFP content of individual Salmonella. We evaluated more than hundred different GFP-constructs but only one single-copy chromosomal P(sifB)-gfp fusion met these criteria and enabled differentiation of weakly and highly infected cells based on total GFP fluorescence. Confocal microscopy of sorted cells confirmed the successful separation of these subpopulations. In addition to isolation of infected cells, our method also enabled enumeration of the subpopulations and phenotypic characterization by staining with antibodies to surface markers. Surprisingly, a small subpopulation of highly infected host cells contained the majority of Salmonella but based on MHC II and ICAM I expression, this subpopulation was not more strongly activated than weakly infected cells. Our method will facilitate future characterization of the different subpopulations and the identification of bacterial and host factors that control Salmonella load and proliferation in vivo.     

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.