Non-selective Pre-enrichment Research Articles

An evaluation of immunomagnetic separation-real-time PCR (IMS-RTiPCR) combined assay for rapid and specific detection of Escherichia coli O157:H7 in raw milk and ground beef

Abstract The aim of this study is to optimize a rapid and specific method for detection of E. coli O157:H7 (EHEC) from food samples with high background flora using an immunomagnetic separation-real-time polymerase chain reaction assay (IMS-RTiPCR). For this, EHEC cells were recovered from raw milk and ground meat samples that were artificially contaminated to the final concentrations of 101, 103 and 105 cfu EHEC/mL or g, after a non-selective pre-enrichment at 35 °C for 8 h following either with or without capturing by micro-sized beads. Then, EHEC cells were identified by RTiPCR. The study was also carried out without any enrichment to evaluate the enrichment efficiency of the assay. By comparing the assay with and without pre-enrichment, we showed that even the usage of specific micro-sized beads did not improve EHEC detection in raw milk and ground beef samples and so gave false negative results unless an 8-h pre-enrichment was applied. Besides, the assay can be completed in less than 9 h with a minimum detection limit of 103 cfu EHEC/mL or g in foods and hence was found to be simple routine-based method that exhibits great potential for implementing as a rapid screening of EHEC in microbial tests as compared to existing techniques.

Evaluación de una Técnica de PCR-Múltiple para la Detección Rápida de Salmonella Typhimurium y Enteritidis en Cuyes (Cavia porcellus) Naturalmente Infectados

El objetivo del estudio fue evaluar la capacidad de detección de Salmonella Typhimurium y Enteritidis mediante el método de la Reacción en Cadena de la Polimerasa (PCR) Múltiple a partir de muestras en pre-enriquecimiento no selectivo (utilizando secuencias blanco de los genes InvA, fliC y prot6E), y hallar su concordancia con el método de detección microbiológica convencional, que consta de pre-enriquecimiento no selectivo, enriquecimiento selectivo, aislamiento en agar diferencial y pruebas bioquímicas. Se analizaron 111 muestras de hígado de cuyes con diagnóstico presuntivo de Salmonelosis, provenientes de Chancay (Lima) y El Mantaro (Junín), Perú. Se llegó a detectar Salmonella Typhimurium por PCR Múltiple en 54% (60/111) de las muestras y en 41% (45/111) por análisis microbiológico. Se encontró una concordancia substancial con un valor de Kappa de 0.64. La prueba de McNemar demostró que los resultados de ambas pruebas son estadísticamente diferentes (p<0.05).

Early Recovery of Salmonella from Food Using a 6-Hour Non-selective Pre-enrichment and Reformulation of Tetrathionate Broth.

Culture based methods are commonly employed to detect pathogens in food and environmental samples. These methods are time consuming and complex, requiring multiple non-selective and selective enrichment broths, and usually take at least 1 week to recover and identify pathogens. Improving pathogen detection in foods is a primary goal for regulatory agencies and industry. Salmonella detection in food relies on a series of culture steps in broth formulations optimized to resuscitate Salmonella and reduce the abundance of competitive bacteria. Examples of non-selective pre-enrichment broths used to isolate Salmonella from food include Lactose, Universal Pre-enrichment, BPW, and Trypticase Soy broths. Tetrathionate (TT) and Rappaport–Vassiliadis (RV) broths are employed after a 24-h non-selective enrichment to select for Salmonella and hamper the growth of competitive bacteria. In this study, we tested a new formulation of TT broth that lacks brilliant green dye and has lower levels of TT . We employed this TT broth formulation in conjunction with a 6-h non-selective pre-enrichment period and determined that Salmonella recovery was possible one day earlier than standard food culture methods. We tested the shortened culture method in different non-selective enrichment broths, enumerated Salmonella in the non-selective enrichments, and used 16S rRNA gene sequencing to determine the proportional abundances of Salmonella in the TT and RV selective enrichments. Together these data revealed that a 6-h non-selective pre-enrichment reduces the levels of competitive bacteria inoculated into the selective TT and RV broths, enabling the recovery of Salmonella 1 day earlier than standard culture enrichment methods.

Cilantro microbiome before and after nonselective pre-enrichment for Salmonella using 16S rRNA and metagenomic sequencing.

BackgroundSalmonella enterica is a common cause of foodborne gastroenteritis in the United States and is associated with outbreaks in fresh produce such as cilantro. Salmonella culture-based detection methods are complex and time consuming, and improvments to increase detection sensitivity will benefit consumers. In this study, we used 16S rRNA sequencing to determine the microbiome of cilantro. We also investigated changes to the microbial community prior to and after a 24-hour nonselective pre-enrichment culture step commonly used by laboratory analysts to resuscitate microorganisms in foods suspected of contamination with pathogens. Cilantro samples were processed for Salmonella detection according to the method in the United States Food and Drug Administration Bacteriological Analytical Manual. Genomic DNA was extracted from culture supernatants prior to and after a 24-hour nonselective pre-enrichment step and 454 pyrosequencing was performed on 16S rRNA amplicon libraries. A database of Enterobacteriaceae 16S rRNA sequences was created, and used to screen the libraries for Salmonella, as some samples were known to be culture positive. Additionally, culture positive cilantro samples were examined for the presence of Salmonella using shotgun metagenomics on the Illumina MiSeq.ResultsTime zero uncultured samples had an abundance of Proteobacteria while the 24-hour enriched samples were composed mostly of Gram-positive Firmicutes. Shotgun metagenomic sequencing of Salmonella culture positive cilantro samples revealed variable degrees of Salmonella contamination among the sequenced samples.ConclusionsOur cilantro study demonstrates the use of high-throughput sequencing to reveal the microbiome of cilantro, and how the microbiome changes during the culture-based protocols employed by food safety laboratories to detect foodborne pathogens. Finding that culturing the cilantro shifts the microbiome to a predominance of Firmicutes suggests that changing our culture-based methods will improve detection sensitivity for foodborne enteric pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0497-2) contains supplementary material, which is available to authorized users.

Automation Highlights from the Literature

Automation Highlights from the Literature

Salmonellaimportance and current status of detection and surveillance methods

Salmonella, a genus within Enterobacteriaceae, remains as an important human pathogen and it has been reported to be the most common food-borne bacterial disease in the world. Although majority of the Salmonella cases are sporadic, outbreaks occur frequently. Salmonella can be associated with many kinds of foods and the presence of Salmonella in ready-to-eat foods is considered significant regardless of the level of the contamination. Therefore isolation is carried out by enrichment culture of a defined weight or volume of the food (normally 25 g). The traditional and time-consuming detection and isolation of Salmonella spp. from food and feed utilizes a multistep protocol with nonselective pre-enrichment, followed by a selective enrichment step, isolation on selective agar media and a preliminary biochemical and serological confirmation. Several rapid methods have been developed to speed up the detection of Salmonella. This paper aims to give an overview of the occurrence and current status of Salmonella detection and surveillance methods.

Rapid detection of Salmonella in milk by combined immunomagnetic separation-polymerase chain reaction assay

During the past few years, milk has presented a risk of Salmonella contamination; it has been implicated as the cause in several outbreaks of salmonellosis. Because conventional detection methods require 5 to 7 d for completion and involve several subcultivation stages followed by biochemical and serological tests, rapid and sensitive methods have been sought, mainly at the DNA level. Therefore, a study including milk samples was conducted to evaluate the performance of a combination of 2 techniques—immunomagnetic separation and polymerase chain reaction (PCR)—for the detection of Salmonella. The 16-, 14-, 12-, 10-, and 8-h nonselective pre-enrichment steps before immunomagnetic separation and the high-pure DNA preparation method before PCR were used in a combined assay. Milk samples, which were found to be Salmonella-negative by a reference method, were first inoculated with Salmonella Enteritidis. Next, the shortest pre-enrichment time that is required for detection of 1 or 10 cfu of Salmonella/mL by combined immunomagnetic separation-PCR assay was found by using 16-, 14-, 12-, 10-, and 8-h incubation periods. The detection limit using a 16-, 14-, or 12-h nonselective pre-enrichment was 1 to 10 cfu/mL. However, the sensitivity decreased to 101 and 102 cfu/mL, respectively, when 10- and 8-h pre-enrichments were used. This assay, in conjunction with a 12-h pre-enrichment, proved to be rapid (overall 16h) and sensitive (1–10 cfu/mL) for the detection of Salmonella in milk samples and promising for routine use in the detection of Salmonella in milk.

Application of magnetic immuno-polymerase chain reaction assay for detection of Salmonella spp. in chicken meats

Since contaminated chicken meats have been the principal foodborne source of the contamination of Salmonella to human beings and cultural detection methods are labor-intensive and time-consuming, a study evaluating the performance of the combination of two techniques that are immunomagnetic separation (IMS) and polymerase chain reaction (PCR) for the detection of Salmonella in chicken meats was conducted. The IMS and PCR assay combines selective extraction of Salmonella by specific antibodies with primer-specific (primer pair based on the sequence of invA gene) PCR amplification. Initially chicken meat samples, in which no Salmonella contamination had been determined by using ISO 6579 reference method, were inoculated with Salmonella Enteritidis culture and subsequently the shortest non-selective pre-enrichment time, that had been needed for the detection of approximately 1 or 10 CFU/mL chicken meat levels of target bacteria by magnetic immuno-PCR assay, was found by using 14, 12, 10 and 8-h periods. In conclusion, it was found that magnetic immuno-PCR assay was able to detect 1–10 CFU Salmonella/25 g chicken meat, after only incorporating a non-selective pre-enrichment period of 12 h. Therefore, an overall 16-h (magnetic immuno-PCR assay in conjunction with 12-h non-selective pre-enrichment) magnetic immuno-PCR assay statistically evaluated as sufficient (p = 0.182 > 0.05) for rapid and sensitive detection of approximately 1–10 CFU Salmonella from 25 g chicken meat samples. Accordingly, 16-h magnetic immuno-PCR assay can be promising for routine use in the detection of Salmonella in chicken meat samples, and it consequently may prevent the risk of Salmonella infections in regard to chicken meats.

The Effect of Preenrichment and Selective Enrichment Media on Recovery of Salmonella Typhi from the Tropical Fruit Mamey

The Bacteriological Analytical Manual (BAM) Salmonella culture method did not detect Salmonella Typhi in mamey, the tropical fruit that was implicated in a 1999 typhoid outbreak. The relative effectiveness of BAM's nonselective preenrichment and selective media for the recovery of S. Typhi from mamey was examined to determine if the BAM's preenrichment/selective enrichment strategy was the cause of the method's failure with this food. The preenrichment media were lactose broth, buffered peptone water (BPW), and universal preenrichment (UP) broth. The selective enrichment media were selenite cystine (SC) broth, tetrathionate (TT) broth, and Rappaport-Vassiliadis (RV) medium. UP broth was significantly more effective (P < 0.05) than either lactose broth or BPW for the recovery of 2 different S. Typhi strains from mamey. Of 120 test portions tested, 105 S. Typhi-positive test portions were recovered using UP broth, whereas only 1 S. Typhi-positive test portion was recovered using BPW, and no S. Typhi-positive test portions were recovered using lactose broth. SC and TT broths were both significantly more effective (P < 0.05) than RV medium. Of 105 S. Typhi-positive test portions, SC broth recovered 80, TT broth recovered 67, and RV medium recovered 9. After the above comparison, an incomplete UP (UPI) broth formulation was found to be significantly more effective (P < 0.05) than the complete formulation (UPC). Of 80 total positive test portions, UPI recovered 71, whereas UPC recovered only 48. The following UP broth formulations were compared to determine if any of the components of the UP broth formulation were inhibitory to S. Typhi: UPC, UP broth without sodium pyruvate (UPS), UP broth without ferric ammonium citrate (UPF), UP broth without MgSO4 (UPM), and UPI. It was found that none of the ingredients were inhibitory to S. Typhi and that, out of 140 total test portions, UPI and UPF, with 108 and 103 positive test portions, respectively, were significantly more effective (P < 0.05) than the other UP broth formulations (82 for UPC, 74 for UPS, and 60 for UPM). Rather than being inhibitory to the growth of S. Typhi, it appears that ferric ammonium citrate enhanced the growth of competitors which suppressed the growth of S. Typhi. These results demonstrate that UPI and UPF are effective for the recovery of S. Typhi from mamey.

Evaluation of an Immunomagnetic Separation–Real-Time PCR Assay for the Rapid Detection of Salmonella in Meat

The aim of this study was the comparison of an immunomagnetic separation (IMS)–real-time PCR assay for the detection of Salmonella with the cultural reference method according to §35 of the German Law on Food and Commodities (LMBG, L 00.00.20:1998). The IMS–real-time PCR assay includes a nonselective preenrichment step, an IMS, DNA extraction, as well as DNA purification followed by hybridization probe–based real-time PCR analysis. An accurate comparability was achieved, because both methods analyzed the same preenrichment. The evaluation was carried out using both artificially and naturally contaminated meat samples. The IMS–real-time PCR assay provides a result after 12 to 13 h. Compared with the reference method and regarding artificially contaminated meat samples, the IMS–real-time PCR assay achieved a specificity of 80% (false-positive rate of 20%) and a sensitivity of 100% (false-negative rate of 0%). The relative accuracy was 94%. The detection limit of both methods was 10 CFU/25 g. The concordance indexκ defines the statistical accordance, was 0.85 and indicated the agreement of both methods on statistical criteria. Compared to the reference method and analyzing naturally contaminated meat samples (n = 491), the IMS–real-time PCR assay showed a specificity of 99.3% (false-positive rate of 0.7%) and a sensitivity of 83.7% (false-negative rate of 16.3%). The relative accuracy was 98%. The concordance index κ had a value of 0.87 and highlighted the statistical agreement of both methods. In conclusion, the IMS–real-time PCR assay is suitable as specific, sensitive, and rapid screening method for the detection of Salmonella from meat.

Validated PCR Assay for the Routine Detection of Salmonella in Food

We developed a rapid and reliable PCR assay with genus-specific primers for the detection of Salmonella in food samples. With these primers, no primer-specific amplicons were detected when challenged with cultures of microorganisms other than salmonellae, and positive results, i.e., Salmonella-specific bands, were obtained with pure cultures of all 125 Salmonella isolates tested, which represented 100 serovars. The PCR assay was optimized using both pure cultures and artificially inoculated food samples. The assay results were compared with those of the Australian standard culture methods, using more than 500 “naturally” contaminated food samples, over a period of 9 years. Food samples were subjected to nonselective preenrichment in buffered peptone water followed by selective enrichment in Rappaport Vassiliadis (RV) broth and mannitol selenite cystine (MSC) broth. A simple sample preparation method was developed based on concentrating bacterial cells from 1 ml of RV or MSC broths. The PCR results were in perfect agreement with the results of the standard culture methods; no false-positive or false-negative results were obtained. However, the PCR assay was extremely rapid, and results could be obtained within 4 h of testing of enrichment broths.

Relative Effectiveness of the Bacteriological Analytical Manual Method for the Recovery of Salmonella from Whole Cantaloupes and Cantaloupe Rinses with Selected Preenrichment Media and Rapid Methods

Soak and rinse methods were compared for the recovery of Salmonella from whole cantaloupes. Cantaloupes were surface inoculated with Salmonella cell suspensions and stored for 4 days at 2 to 6° C. Cantaloupes were placed in sterile plastic bags with a nonselective preenrichment broth at a 1:1.5 cantaloupe weight-to-brothvolume ratio. The cantaloupe broths were shaken for 5 min at 100 rpm after which 25-ml aliquots (rinse) were removed from the bags. The 25-ml rinses were preenriched in 225-ml portions of the same uninoculated broth type at 35° C for 24 h (rinse method). The remaining cantaloupe broths were incubated at 35° C for 24 h (soak method). The preenrichment broths used were buffered peptone water (BPW), modified BPW, lactose (LAC) broth, and Universal Preenrichment (UP) broth. The Bacteriological Analytical ManualSalmonella culture method was compared with the following rapid methods: the TECRA Unique Salmonella method, the VIDAS ICS/SLM method, and the VIDAS SLM method. The soak method detected significantly more Salmonella-positive cantaloupes (P < 0.05) than did the rinse method: 367 Salmonella-positive cantaloupes of 540 test cantaloupes by the soak method and 24 Salmonella-positive cantaloupes of 540 test cantaloupes by the rinse method. Overall, BPW, LAC, and UP broths were equivalent for the recovery of Salmonella from cantaloupes. Both the VIDAS ICS/SLM and TECRA Unique Salmonella methods detected significantly fewer Salmonella-positive cantaloupes than did the culture method: the VIDAS ICS/SLM method detected 23 of 50 Salmonella-positive cantaloupes (60 tested) and the TECRA Unique Salmonella method detected 16 of 29 Salmonella-positive cantaloupes (60 tested). The VIDAS SLM and culture methods were equivalent: both methods detected 37 of 37 Salmonella-positive cantaloupes (60 tested).

CONCURRENT DETECTION OF ESCHERICHIA COLI O157:H7 AND SALMONELLA FROM A SINGLE ENRICHMENT IN 24 H

ABSTRACT The objective of this study was to determine if a single assay protocol could result in the concurrent detection of Escherichia coli O157:H7 and Salmonella from a single sample grown in a single enrichment in 24 h. Twenty‐five and 375 g of ground beef nonfat dry milk, and dry pet food samples were seeded with low (10 cfu/sample) and high (100 cfu/sample) levels ofE. coli O157:H7 and Salmonella cultures and incubated at 35 and 41C for 18 h for nonselective preenrichment. Incubated samples were analyzed by immunomagnetic separation (IMS) following a 6 h incubation for selective enrichment at 37C using M‐broth and enzyme linked immumosorbent assay (ELISA). Depending on the food samples and the inoculation level, the minimum concurrent detection level of E. coli O157:H7 and Salmonella was &lt;1 cfu/g in the samples at the competitor flora level of 105 cfu/g or less in ground beef samples, but in other cases of higher competitor loads and low target inoculations E. coli O157:H7 could not be detected in the presence of the Salmonella.

Comparison of Enrichment Methods for Recovery and Chick Infectivity of Chlorine-lnjured Salmonella enteritidis

In recent years, several preenrichment media have been shown to be effective for use in the recovery of sublethally injured Salmonella organisms. Selective enrichment without preenrichment has resulted in a lower recovery of organisms, particularly with regard to injured or stressed salmonellae. The present experiments compared the ability of nonselective preenrichment followed by selective enrichment or direct selective enrichment alone to recover chlorine-injured Salmonella organisms. Additionally, the Salmonella detection limits of the two enrichment methods were compared with minimal infectious dose in neonatal chicks. In three experiments, Salmonella enteritidis cells were exposed to chlorine for specific times and subsequently cultured by using preenrichment followed by selective enrichment or selective enrichment alone. Simultaneously, neonatal chicks were orally challenged with S. enteritidis cells from each exposure time to chlorine. The results indicated a marginal, but significantly (P < 0.05) higher level of recovery of sublethally injured salmonellae by using nonselective preenrichment followed by selective enrichment, as compared to selective enrichment alone. Interestingly, both culture methods were capable of detecting injured S. enteritidis cells at levels incapable of infecting neonatal chicks.

Injury, resuscitation and detection of Listeria spp. from frozen environments

The ability of Listeria spp. to be reversibly freeze-injured was determined and methods for detection of freeze-injured Listeria were evaluated. Strains of Listeria monocytogenes and Listeria innocua tested showed no significant variation as to the extent of injury sustained during storage for 24 h at -9 to -11°C. Average 24 h injury ranged from 44-64% for L. monocytogenes strains and 41-54% for L. innocua strains. The most substantial increase in injury was seen in the first 24 h and injury remained constant or increased slightly throughout the 14 day period. Freeze injury was reversible in all Listeria strains tested when trypticase soy broth and Listeria repair broth (LRB) were used as repair media. In order to determine if nonselective pre-enrichment followed by selective enrichment using LRB, resulted in increased detection of Listeria existing naturally in processing environments, samples were obtained from two dairy and one meat processing plant. Use of nonselective pre-enrichment followed by selective enrichment using LRB did not enhance recovery of Listeria from frozen environments. This may be attributed to a limited amount of freeze-injury occurring in a naturally existing population of Listeria.

Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry.

The use of fluorescently-labelled monoclonal antibodies, with detection by multi-parameter flow cytometry, was investigated for the rapid detection of salmonellas in pure cultures. Accurate detection of specific Salmonella serotypes was demonstrated down to levels of below 10(4) cells ml-1 (within 30 min) and 1 cell ml-1 (after 6 h non-selective pre-enrichment). This level of sensitivity was attained even in the presence of high levels of other bacterial species that would otherwise have interfered with the results. With combinations of different antibodies, each with a unique fluorescent label, simultaneous analysis for two species was possible.

Recovery of Salmonella enteritidis From Inoculated Pools of Egg Contents

To determine whether Salmonella enteritidis (SE) could be efficiently detected in pools of egg contents, eggs were inoculated with small numbers of SE and the contents of 10 or more eggs were mixed together. After these pools were incubated, samples were transferred to culture media for isolation and identification of SE. Incubation (at either 25 or 37°C) of inoculated egg pools for at least 4 d before culturing resulted in a significant increase in the frequency of recovery of SE. Following inoculation of 10-egg pools with approximately 50 CFU, SE was recovered from 11% of pools before incubation and from 94% of incubated pools. Following inoculation of 10-egg pools with approximately 5 CFU, SE was recovered from none of the pools before incubation and from up to 72% of the incubated pools. Increasing the size of the pools (from 10 to 30 eggs) did not significantly affect the frequency of isolation of SE, but reducing the sample volume transferred from incubated egg pools to culture media (from 20 to 2 ml) led to a significant decrease in SE recovery. At the lower (2 ml) transfer volume, the frequency of recovery of SE was not improved by nonselective preenrichment of samples.

Evaluation of enrichment procedures for recovering Listeria monocytogenes from dairy products

Six different enrichment media and five selective plating media were compared for their suitability for the recovery of Listeria monocytogenes from dairy products. These included media used to test milk products by the U.S. Food and Drug Administration (FDA) and the U.S. Centers for Disease Control (CDC), and media developed by the U.S. Department of Agriculture (USDA) for testing meat and poultry products. Test samples included naturally contaminated goat's milk, cultured milk products and ice cream manufactured with L. monocytogenes, and unpasteurized milk inoculated with heat- and freeze-injured cells of L. monocytogenes. Generally, the media and two-stage enrichment protocol developed by the USDA, with plating of samples after two consecutive 24-h incubation periods, yielded better recoveries than all other enrichment media incubated for 24 h. A modified USDA procedure, incorporating nonselective pre-enrichment of samples by omitting acriflavine and nalidixic acid from the primary USDA enrichment broth, and transfer of a larger volume of the initial culture broth to the secondary enrichment media, significantly increased recoveries of low numbers of sublethally stressed L. monocytogenes. Prolonged incubation of samples in the FDA enrichment broth, for 7 days, did not consistently improve recoveries over the initial 24-h incubation time of the medium. The selective plating medium developed by the USDA, lithium chloride-phenylethanol-moxalactam agar, was the most effective plating agar for isolation of L. monocytogenes following enrichment of samples in any broth culture, and increased recoveries of L. monocytogenes by 19–40% compared with other selective agar media tested.

Effect of Various Media and Incubation Conditions on Recovery of Inoculated Salmonella from Poultry Feed

Samples of commercially pelleted poultry feed (30 g) were inoculated with nalidixic acid-resistant marker strains of Salmonella heidelberg or S. montevideo at levels of 1 (low), 20 (medium), or 40 (high) cells/g of feed, then 100 ml of either a nonselective preenrichment medium (lactose broth) or a selective enrichment medium [selenite cystine (SC) or tetrathionate brilliant green (TT) broth] was added and incubated at either 37 or 43 C for 24 or 48 hr in different experiments. Four selective plating media [MacConkey with 100 ppm nalidixic acid, brilliant green (BG) sulfa, modified BG, bismuth sulfite] were then streaked. MacConkey agar with nalidixic acid served as a control to which the other three plating media were compared. After 24 hr incubation at 37 C, colonies with characteristics typical of Salmonella were selected from the plates and examined biochemically and serologically to determine if they were the marker organism.In this study, preenrichment was counterproductive, because the marker organisms were recovered much more often with direct enrichment in SC or TT broth than with preenrichment in lactose broth. The TT broth produced 135 positive recoveries, whereas only 88 positive recoveries were made with SC. No differences were observed between 37 and 43 C or 24 and 48 hr incubation of the selective enrichment media. Overall efficiency of the plating media for Salmonella recovery from feed, when compared to the control (MacConkey), was BG sulfa, 65%; modified BG, 64%; and bismuth sulfite, 40%. The most effective enrichment broth-plating medium combination was TT-BG sulfa, yielding 86% positive recoveries when compared to the control.