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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.36 no.2 São Paulo Apr./June 2005

https://doi.org/10.1590/S1517-83822005000200009 

FOOD MICROBIOLOGY

 

Evaluation of three enrichment broths and five plating media for Salmonella detection in poultry

 

Avaliação de três caldos de enriquecimento e cinco meios de cultura para detecção de Salmonella em carcaças de frango

 

 

Vera Lúcia Mores RallI,*; Ricardo RallI; Lina Casale AragonI; Márcia Guimarães da SilvaII

IDepartamento de Microbiologia e Imunologia, Universidade Estadual Paulista, Botucatu, SP, Brasil
IIDepartamento de Patologia, Universidade Estadual Paulista, Botucatu, SP, Brasil

 

 


ABSTRACT

We evaluated the effectiveness of Selenite Cystine (SC), Tetrathionate Brilliant Green (TBG) and Rappaport Vassiliadis (RV) broths for Salmonella isolation. We also tested three classic plating media, Salmonella-Shigella Agar (SS), Brilliant Green Agar (BGA), Xylose Lysine Desoxycholate Agar (XLD) and two chromogenic agars, Rambach (RA) and CHROMagar Salmonella (CAS). Among 100 poultry carcasses, 29 were positive for Salmonella using all plating media combined. RV broth (69%) and TT broth (58.6%) were more effective than SC broth (24.1%). The chromogenic media gave better results than the classic ones with less false-positive colonies. The most effective isolation medium was CHROMagar, where Salmonella was identified in 23 (79.3%) of the 29 positive samples, followed by Rambach (48%). Positivity for Salmonella using classic media was 13.8% for BGA, 27.6% for SS and 34.5% for XLD.

Key words: Salmonella, poultry, plating media, selective enrichment


RESUMO

A eficiência dos caldos selenito cistina (SC), tetrationato verde brilhante (TBG) e Rappaport Vassiliadis (RV) foi avaliada quanto ao isolamento de Salmonella. Também foram testados três meios clássicos de isolamento, ágar Salmonella-Shigella (SS), ágar Verde Brilhante (VB) e ágar xilose lisina desoxicolato (XLD) e dois meios cromogênicos, Rambach (RA) e CHROMagar (CAS). Entre 100 carcaças de frango examinadas, 29 foram positivas para Salmonella usando todos os meios combinados. Os caldos RV (69%) e TT (58,6%) foram mais eficientes que o SC (24,1%). Os meios cromogênicos mostraram melhores resultados do que os clássicos na detecção de Salmonella e apresentaram uma quantidade menor de colônias falso-positivas. O meio cromogênico mais eficiente foi o CAS, que detectou Salmonella em 23 das 29 amostras positivas (79,3%), seguido pelo RA (48%). Entre os meios clássicos, a detecção foi de 13,8% para VB, 27,6% para SS e 34,5% para XLD.

Palavras-chave: Salmonella, aves, meios de isolamento, enriquecimento seletivo


 

 

INTRODUCTION

Foodborne diseases are an important public health problem. Salmonella infects approximately 1.4 million people, resulting in several hundred deaths per year just in the United States. A wide variety of selective media have been developed to isolate this microorganism, but not one is considered perfect. Selective enrichment broths lead to increased salmonella numbers because contain inhibitory compounds that limit non-salmonella microorganisms. At present, enrichment broths containing selenite, and both brilliant green and malachite green are recommended (5). Also currently Rappaport-Vassiliadis broth (RV) medium is recommended for Salmonella recovery from low and highly contaminated foods, while tetrathionate broth (TT) incubated at 35ºC is indicated for foods with low microbial load, and at 43ºC for high loads. Traditional plating media are based on lactose fermentation and hydrogen sulfide production. Most are nonspecific and expensive, and the identification is time consuming. Since 1990's, several chromogenic media have been developed to detect Salmonella, such as Rambach (22), SM-ID (21), CHROMagar (9), ABC Medium (19), Chromogenic Salmonella esterase agar (6), and Rainbow Salmonella Agar (15). They are based on a combination of biochemical characteristics and are highly specific.

The objective of this study was to evaluate the effectiveness of three enrichment broths and five plating agars for Salmonella isolation from naturally contaminated poultry carcasses.

 

MATERIALS AND METHODS

Samples

One hundred refrigerated poultry carcass samples were collected from supermarkets, butcheries, and hospital kitchens over a one year period.

Procedures

Two hundred and twenty five mL of buffered peptone broth (Oxoid) and 25g of poultry carcass were homogenized in a Stomacher Lab Blender 400 for one minute and incubated for 18-20h at 37ºC (pre-enrichment). After incubation, 0.1 mL was transferred to 10 mL of Rappaport-Vassiliadis broth (RV) (Oxoid) and 1mL to 10 mL of Tetrathionate Brilliant Green broth (TT) (Difco); both were incubated at 42ºC for 24h. Selenite broth (SC) (10 mL) (Oxoid) was inoculated with 1mL of the pre-enrichment broth and incubated at 37ºC for 24 h. After incubation at 37ºC or 42ºC, a loopfull of each broth was plated onto Rambach agar (Merck), CHROMagar Salmonella, SS Agar (Salmonella-Shigella) (Oxoid), Brilliant Green Agar (BGA) (Oxoid) and Xylose Lysine Desoxycholate Agar (XLD) (Oxoid). After incubation, five typical colonies from each agar plate were submitted to biochemical tests using TSI agar (Difco) and API-20E (Biomérieux). The colonies were also submitted to serological tests using polyvalent somatic and flagellar antisera (Probac do Brasil).

Statistical evaluation

The Cochran test was used to compare the proportions of dependent samples from the enrichment media (11); the Z test was used to compare the results from media with or without chromogenic properties (25). The significance level for all tests was 5%.

 

RESULTS AND DISCUSSION

Twenty nine samples were positive for Salmonella. The pathogen was detected in 7 samples (24.1%) when selenite cystine broth (SC) was used. With tetrathionate (TBG) and Rappaport-Vassiliadis (RV), the isolation rate was higher: 58.6% (17 samples) and 69% (20 samples), respectively. The difference between results in TBG and RV was not significant; however both were considered more effective than SC.

Several authors (3,12,14,20,24) observed that RV is the most effective enrichment broth for Salmonella. However, in our study the difference between RV and TT was not significant. Vassiliadis (24) tested 2,000 samples of meat products, pig faeces, and sewage and observed that 17% were positive for Salmonella with TT, and 25% with RV. Similar results were observed by Pietzsch (20) who analyzed samples of faeces, pork, and chicken skin and observed 48% and 92% positivity with TT and RV, respectively. Beckers et al. (3) tested 590 samples of dehydrated vegetables, chicken, egg, pepper, and ground meat and observed that 63% were positive for Salmonella when RV was used and 47% when TT was used. June et al. (14) and Hammack et al. (12) also found that RV and TT at 42ºC were better than SC and TT at 35ºC for isolation of Salmonella from fresh meat and highly contaminated products.

June et al. (14) tested three enrichment procedures, RV at 42ºC, TT at 35º and 42ºC and SC at 35ºC, to assess their effectiveness in recovering Salmonella from artificially contaminated oysters, frog paws, mushrooms, shrimps and non-contaminated poultry. Among 1,125 samples, the positivity for Salmonella was 36.3% with RV, 27.5% and 32.7% with TT at 35 and 42ºC, respectively, and 29.7% with SC.

Blivet et al. (4) also observed that SC was less effective than RV. Testing chicken, egg, and turkey samples, they found Salmonella in 97.6% of the positive samples using RV, and only 42.2% using SC. However, they noticed that the SC broth was able to detected low numbers (10 to 50 CFU/mL) of some serotypes of Salmonella, such as S. Gallinarum, S. Pullorum, S. Typhi and S. Paratyphi.

In this study, the lowest recovery of Salmonella using SC broth agrees with results of other studies (13). Bailey et al. (2) compared the efficiency of SC and TT for the isolation of Salmonella from artificially and naturally contaminated food samples. When the authors analyzed contaminated ground meat and hot dogs, SC was more effective for ground meat while TT was more effective for hot dogs. This can be explained by the microbiota found in these foods. In the 70's, Fagerberg and Avens (8) reported that SC was better for certain serotypes of Salmonella, while TT was more efficient for others. However, Cox and Mercuri (7) observed that TT was more toxic when the concentration of microorganisms was low. Therefore, SC can be the most effective broth for certain types of food, where the numbers of Salmonella are low.

Arroyo and Arroyo (1) analyzed 264 chicken and sheep samples and found Salmonella in 83 (31.4%). They used TT at 37ºC, RV at 42ºC and SC at 37 and 43ºC, and observed that SC gave better results than TT or RV, regardless the incubation temperature.

In our study, the chromogenic media were more efficient and presented fewer false positives than the classic Salmonella media. Fig. 1 shows that the most effective plating medium was CHROMagar which detected Salmonella in 23 out of 29 positive samples (79.3%), followed by Rambach (48.3%). XLD was the best classic medium (34.5%), followed by SS (27.6%), and BGA (13.8%). These results are similar to those reported by Rhodes and Quesnel (23) and Moringo et al. (16).

 

 

Our data also showed that CHROMagar (CAS) was better than Rambach (RA); This is in agreement with Narquet and Roupas (17,18), who observed that CAS was able to detect more positive samples than Rambach.

 

 

Gaillot et al. (9) tested the efficacy of CAS and Hecktoen Enteric Agar (HE) for isolation of Salmonella from 508 stool samples. Twenty samples were Salmonella positive: CAS detected 19 and HE, 16. The number of false-positive results was lower with CAS than with HE.

In classic media, Salmonella,Proteus and Citrobacter present similar biotypes: they are negative for lactose fermentation and produce H2S. Since Proteus and Citrobacter are commonly found in foods, the number of false-positive colonies in classic media may be very large.

False-positive colonies can also occur in chromogenic media: only 21% of the 157 characteristic colonies in RA were confirmed as Salmonella. This was due to the large number of Proteus and Citrobacter in the samples, which can also produce red coloration in this media. According to Rambach (22), these colonies should be blue; however red colonies were also observed by Garrick and Smith (10) and Gaillot et al. (9). In this study, Proteus colonies presented the same pale-yellow color. The lowest number of false positive colonies was observed in CHROMagar where 82 from 149 were positively identified as Salmonella. In this medium, characteristic Salmonella colonies had a light violet coloration, while other enterobacteria, including Proteus and Citrobacter, presented a blue color. False positive colonies that were not Proteus or Citrobacter were mainly Pseudomonas aeruginosa or other small Gram negative rods. Gaillot et al. (9) also observed that Pseudomonas aeruginosa and Aeromonas hydrohila can grow in this medium; the small rods seen in our study may belong to these two species.

Therefore, for Salmonella to be detected efficiently, besides two enrichment broths incubated at different temperatures, a chromogenic plating medium should also be used.

 

ACKNOWLEDGEMENTS

We thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support.

 

REFERENCES

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Submitted: November 13, 2002; Returned to authors for corrections: June 06, 2003; Approved: June 15, 2005

 

 

* Corresponding Author. Mailing address: Departamento de Microbiologia e Imunologia, Instituto de Biociências, Universidade Estadual Paulista, Caixa Postal 510, 18618-000, Botucatu, São Paulo, Brasil. Tel. (+5514) 3811-6240, Fax (+5514) 3815-3744. E-mail: vlmores@ibb.unesp.br

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