Comparison of five culture methods for Salmonella isolation from swine fecal samples of known infection status

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1 620 Brief Research Reports J Vet Diagn Invest 20: (2008) Comparison of five culture methods for Salmonella isolation from swine fecal samples of known infection status Brenda C. Love, 1 Marcos H. Rostagno Abstract. The current study was conducted to evaluate 5 bacteriologic culture methods (methods 1, 2, 3, 4, and 5) for recovery of Salmonella enterica from swine feces, both for sensitivity of detection (ability to recover Salmonella from a positive sample) and for specificity (not to inadvertently identify an organism as a Salmonella species in a negative sample). Fifty-six negative samples and 46 positive samples were processed using each of the 5 methods, which differed primarily in the combinations of enrichment media used. All negative samples were negative for Salmonella when cultured by all 5 methods (100% specificity). Two of the methods (methods 1 and 4) resulted in the recovery of significantly less (P, 0.05) Salmonella when compared with the remaining 3 methods (methods 2, 3, and 5). No one method was successful in recovering Salmonella from all positive samples, although recovery with method 2 was statistically similar to the total number of positive samples analyzed (42 vs. 46 Salmonella-positive samples, P. 0.05). This study shows that culture methods differ significantly in their performance regarding the isolation of Salmonella from swine fecal samples. Key words: Culture methods; Salmonella; sensitivity; swine. <!?show "fnote_aff1"$^!"content-markup(./author-grp[1]/aff./author-grp[1]/dept-list)> From the Oklahoma Animal Disease Diagnostic Laboratory, Department of Veterinary Biosciences, Oklahoma State University, Stillwater, OK (Love), and the U.S. Department of Agriculture, Agricultural Research Service, Livestock Behavior Research Unit, West Lafayette, IN (Rostagno). 1 Corresponding Author: Brenda C. Love, Oklahoma Animal Disease Diagnostic Laboratory, Department of Veterinary Biosciences, Oklahoma State University, PO Box 7001, Stillwater, OK brenda.love@okstate.edu There has been increasing concern in recent years over the human health risk posed by microbial pathogens in the food supply. Food-borne infections constitute a significant cause of morbidity and mortality in human populations, and infections caused by Salmonella enterica, in particular, have been a major focus of concern. 11 Because of the public health risk associated with consumption of pork from infected pigs, several countries (most notably, Denmark) have instituted surveillance programs to predict the risk of

2 Brief Research Reports 621 carcass contamination at the time of slaughter. 1,13 Additionally, the European Commission is adopting the Danish protocol for preharvest testing of swine for S. enterica as mandatory in This will increase the need for all pork-producing countries, including the United States, to enhance their monitoring and surveillance programs for prevalence detection of S. enterica infection of swine. Preharvest estimates of S. enterica prevalence are most commonly based on traditional culture methods, 7,10,19 despite their lack of sensitivity and expediency. However, there is no single method for culture of S. enterica. In fact, there are probably more techniques and methods for culturing S. enterica than for any other bacterium. This fact was clearly illustrated in a nationwide survey of methods used for culture of S. enterica from poultry samples. 20 In the reported survey, no 2 participants used identical culture methods, and variation occurred in enrichment procedures, growing media, incubation temperatures and times, and multiplicity of culture attempts per sample. Studies performed to determine the sensitivity of various culture methods on different substrates have shown that certain methods perform better with specific substrates. Thus, the use of culture methods appropriate for recovery of Salmonella from food, feed, or environmental sources may not be optimal for recovery from feces. 6,17 Likewise, culture methods for recovery of Salmonella from a given substrate may be species specific. Several recent studies have focused on recovery of S. enterica from naturally infected swine. 3 5,8,9,12,14 16 In general, these studies show that the more you look, the more you find and that no one culture method is optimal for recovery of all serotypes of S. enterica. These studies used fecal samples from herds known to be infected; however, the status of the individual animals at the time of collection of the sample was unknown. As a result, these studies compared the relative sensitivity of isolation between cultural methods. Within an infected herd, the prevalence of S. enterica shedding can vary dramatically over time. 7 As a result, the true status of an individual fecal sample is unknown until at least 1 cultural method shows it to be positive. Consequently, the true diagnostic sensitivity of any particular method cannot be determined. Determining the diagnostic sensitivity of the most commonly applied culture methods using fecal samples from animals known to be shedding organisms at the time of collection or known to be free of infection constitutes a fundamental and necessary step toward establishing parameters and reference methods. A side-by-side evaluation of culture methods for sensitivity and specificity in preharvest food safety investigations will facilitate the standardization of methods, enabling the comparison of results obtained in different laboratories and countries. Additionally, future investigations can be expanded to include additional methods of S. enterica detection (including noncultural methods) and comparison of performance between protocols in various laboratories. Analysis of studies that determine the prevalence of Salmonella infection, based on cultural methods, can then be accomplished with a greater assurance that the diagnostic sensitivity of the method used did not affect the perceived prevalence rate. Therefore, the objective of the current study was to determine the diagnostic sensitivity of commonly applied culture methods for the isolation of S. enterica from swine fecal samples of known infection status. Fecal samples (10 g) from swine known to be shedding at least 1 serotype of S. enterica (n 5 46) and from swine shown previously to be free of S. enterica infection (n 5 56) were used in the present comparative study. To make the comparison more realistic and to account for the common natural occurrence of a wide range of serovars (in the positive samples), pooled fecal samples were collected from several abattoir holding pens that had recently held market swine (for 2 to 4 hr before slaughter) from many different farms. To get as much diversity as possible of serovars, each pooled sample was composed of 6 to 8 individual samples collected from multiple pens. This sampling approach was demonstrated to be successful in obtaining Salmonella-positive fecal samples. 16 The known Salmonella-negative samples were collected directly from the rectum of individual finishing pigs from a Salmonella-free herd (continuously monitored). All samples (i.e., positive and negative) were processed according to a set of 5 culture methods that have been commonly used in epidemiologic investigations and other similar comparative studies 3,4,7,9,13 to isolate S. enterica from swine feces. A flow chart summarizing the 5 culture methods is shown in Figure 1. All microbiological media used in the current study were purchased from a commercial source, a and any preparation of media was carried out according to the manufacturer s directions. Identification of suspect colonies was performed by traditional biochemical reactions, including appropriate reactions on xylose-lysine-tergitol- 4 (XLT4), brilliant green with novobiocin (BGN), MacConkey agar plates, triple sugar iron/lysine iron agar (TSI/LIA) reactions, agglutination using polyvalent Salmonella Oantisera, b and API 20E strips. c Each sample, whether positive or negative, was processed by all 5 culture methods evaluated. Individual samples were thoroughly homogenized and divided in 5 10-g aliquots, which were individually processed for the isolation of Salmonella using each of the 5 culture methods. Data analysis included comparison of proportions with McNemar s chi-square test (statistical significance level for inferences of P, 0.05), determination of agreement between methods (using the kappa statistic), and estimation of diagnostic sensitivity for each method. 18 The 5 culture methods used employed different combinations of nonselective enrichment media (Gram-negative [GN] broth, buffered peptone), selective enrichment media (Rappaport-Vassiliadis [RV] broth, tetrathionate broth [TTB]), and selective/differential agar plates (XLT4, modified semisolid RV agar [MSRV]). Although most incubations were conducted at 37uC, a temperature at which most enteric bacteria grow well, in some methods 42uC was used as an additional selective process.

3 622 Brief Research Reports Figure 1. Flow chart outlining the 5 culture methods used to recover Salmonella from swine fecal samples. All of the Salmonella-negative fecal samples (56/56) were negative by all 5 culture methods applied. None of the culture methods was successful in the recovery of Salmonella from all 46 positive fecal samples analyzed. The diagnostic sensitivity and frequency of Salmonellapositive samples detected by each culture method evaluated is summarized in Table 1. Individually, methods 1 and 4 performed poorly (compared with methods 2, 3, and 5), resulting in significantly lower recovery of Salmonella (P, 0.05), whereas methods 2 and 5 performed best, although they were not able to recover Salmonella from all positive samples. Combined results

4 Brief Research Reports 623 Table 1. Frequency of Salmonella-positive swine fecal samples and relative diagnostic sensitivity of 5 bacteriologic culture methods. Method Frequency* Sensitivity (%) 1 3/46 a /46 b,d /46 c /46 a /46 b 80.4 Any ( standard ) 46/46 d 100 * Different superscript letters indicate statistically significant difference between proportions (P, 0.05). of the culture methods applied are summarized in Table 2. Although the majority of the known positive samples analyzed (82.6%) were correctly identified as Salmonella positive by multiple culture methods, only when combining results of methods 2 and 3 or 2 and 5 were all 46 known Salmonella-positive samples correctly identified. Overall, the agreement between culture methods to identify Salmonella-positive samples was lower than expected. The highest agreement was found between culture methods 2 and 5 (73.2%) and methods 3 and 5 (60.5%), whereas all other agreements between culture methods evaluated were below 40%. The current study shows that there can be significant differences in the ability to recover Salmonella from swine fecal samples when different culture methods are employed. Furthermore, differences in sensitivity of detection were found between very similar culture methods. In general, methods that included preenrichment broths (i.e., nonselective media in the first step of the recovery process) did not perform adequately. Recent studies have shown results consistent with this study. On the other hand, superior recovery of Salmonella from swine feces has been demonstrated by using RV or MSRV 15 with additional modifications to the culture protocol, such as addition of novobiocin to preenrichment or enrichment steps, enhancing the performance of these media. Culture methods may preferentially recover specific serotypes of Salmonella, leading to biased assessment of their performance based on the resident serotypes present in the given fecal samples. 8,16 In the present study, the only culture method with a satisfactory recovery rate of Salmonella from naturally contaminated swine fecal samples was method 2 (statistically similar to the defined standard ; P. 0.05), which included a primary enrichment in the first step of the recovery process with tetrathionate broth. This observation demonstrates the importance of suppressing competitor bacteria in the original sample to allow Salmonella to grow to the level of detection by the plating media applied. Preenrichment is usually recommended for food and environmental samples, which likely have low numbers of Salmonella that may have been stressed by thermal or osmotic shock or by freezing and thawing. However, results of the Table 2. Frequency of Salmonella-positive fecal samples, based on different combinations of results from all culture methods evaluated. Salmonella-positive samples, based on Frequency % General combinations Any 1 method 8/ Any 2 methods 20/ Any 3 methods 16/ Any 4 methods 2/ Any 5 methods 0/46 0 At least 1 method ( standard ) 46/ Specific combinations Methods 1 and 2 42/ Methods 1 and 3 23/46 50 Methods 1 and 4 3/ Methods 1 and 5 37/ Methods 2 and 3 46/ Methods 2 and 4 42/ Methods 2 and 5 46/ Methods 3 and 4 22/ Methods 3 and 5 38/ Methods 4 and 5 37/ present study suggest that preenrichment of heavily contaminated samples, such as feces, may be counterproductive because it allows for an overgrowth of other bacteria that multiply faster than Salmonella. Moreover, it has been shown that different Salmonella serotypes are usually recovered from the same samples processed by different culture methods, 16 which may be due to the differential growth rate between the different serotypes within the same sample. Although Salmonella isolates recovered in the current study were not serotyped, different culture methods often resulted in the recovery of different serogroups of Salmonella from a single sample (data not shown). It must be considered that the samples used in the current study originated from animals not clinically ill (i.e., healthy carriers of Salmonella), and it cannot be assumed that the methods would perform similarly when used on fecal samples from clinically ill animals (i.e., clinical salmonellosis). However, this study has a direct impact on the ability to recognize clinically normal animals that are shedding Salmonella speciesastheyenter the food chain. If the United States begins to implement culture-based, preslaughter surveillance for Salmonella species, as other countries have done, the outcome of culture could be greatly skewed by the use of different culture methods. Based on the results reported in the present study, the recommended culture method for the recovery/isolation of Salmonella from swine fecal samples consists of inoculating 10 g of feces into 100 ml of tetrathionate broth and incubating at 37uC for 48 hr, subculturing 10 ml of the tetrathionate broth into 100 ml RV broth and incubating at 37uC for 24 hr, then inoculating XLT4 agar plates with 10 ml of the RV broth and incubating 24 hr at 37uC before screening for suspect Salmonella colonies. Although the use of multiple culture methods in parallel also provides

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