OBJETO

raw vegetables/produce, most likely occurs during processing, including irrigation, harvesting, and hand packaging. Means to control microbial pathogen contamination may involve chemical treatment or irradiation. Although public attention has been drawn to the control of other microbial pathogens, such as Salmonella and E. coli O157:H7, foods potentially contaminated with Shigella could also be successfully treated with ionizing radiation. Debate over whether there is an adverse effect on the “taste” of the raw commodity after irradiation or over the cost of imple-menting irradiation on a larger scale may lessen the appeal of this process to reduce the number of shigel-lae in foods. Irradiation of foods is supported by U.S. government agencies, such as the FDA (FDA’s Center for Food Safety and Applied Nutrition; “Food Irradiation: a Safe Measure,” http://vm.cfsan.fda.gov/

~dms/opa-bckg.html) and the CDC (http://www.cdc.

gov/ncidod/dbmd/diseaseinfo/foodirradiation.htm), and at the international level by the Codex Alimen-tarius Commission (Codex, 2003a, 2003b; www.

Codexalimentarius.net).

In the United States and other countries (e.g., Canada [http://www.foodproductiondaily.com /news/

ng.asp?id=28788-canada-extends-food]), the appli-cation of food irradiation is slowly being accepted by consumers. In contrast, in Europe, acceptance is moving more slowly. Irradiating foods is an accepted

as S. dysenteriae type I, are unable to grow on highly selective Salmonella-Shigella medium.

Media containing chromogenic or fl uorogenic indicators have been applied to isolating and detec-tion regimens for a number of pathogens, notably E.

coli O157:H7 and Salmonella spp. As for Shigella, one medium containing a chromogenic indicator has been tested and should be commercially avail-able in the near future. In that study chromogenic agar was used with tomatoes artifi cially contami-nated with S. boydii and S. sonnei (Warren et al., 2005). However, this medium fares poorly with foods having a high number of indigenous microbial populations.

Confi rmation is usually performed using bio-chemical tests, either automated or commercially available biochemical strips. Suspected colonies that are gram-negative, nonmotile rods are inoculated onto lysine iron or Kliger iron agar and incubated 18 to 24 hours at 37C. Shigella spp. produce alkaline (red) slants, acid (yellow) butt, no H2S, and no gas, with the exception of S. fl exneri serotype 6, on these agars. Similar to other enteric bacteria, Shigella spp.

are oxidase negative, ferment glucose, and, except for S. dysenteriae type 1, are catalase positive. Further biochemical characterizations show that Shigella spp.

are negative for H2S production, phenylalanine deam-inase, and sucrose and lactose fermentation (although S. sonnei may after a long period of incubation); do not utilize citrate, acetate, potassium cyanide, malonate, inositol, adonitol, and salicin; and lack lysine decarboxylase. Shigellae are negative for the Voges-Proskauer test (S. sonnei and S. boydii sero-type 13 are positive); however, all shigellae are methyl red positive and are unable to produce acid from glu-cose and other carbohydrates (acid and gas produc-tion occurs with S. fl exneri serotype 6, S. boydii serotypes 13 and 14, and S. dysenteriae 3). One Shi-gella sp., S. dysenteriae, is catalase negative and has ornithine decarboxylase activity.

These biochemical tests and those presented in several laboratory protocols aim to differentiate Shigella spp. from E. coli and also to distinguish Shigella spp. from each other. Growth on Chris-tensen citrate, sodium mucate, or acetate agar is one characteristic that discriminates between E. coli and Shigella spp.; shigellae are unable to utilize citrate, acetate, or mucate as a sole carbon source. Other biochemical tests are used to identify the serotypes of Shigella. The ability to utilize mannitol, dulcitol, xylose, rhamnose, raffi nose, glycerol, and indole and the presence of ornithine decarboxylase and o-nitrophenyl- -d-galactopyranosidase have been used to physiologically discriminate between Shigella spp.

regulatory departments and agencies have imple-mented programs to test imported and domestic pro-duce for the presence of select bacterial agents as a means to obtain baseline data and to take regulatory action when warranted. A survey of how extensive this problem is and how the U.S. FDA is addressing this issue has been published (Beru and Salsbury, 2002; www.cfsan.fda.gov/~dms/ prodact.html).

Discriminative Detection Methods for Confi rmation and Trace-Back of

Contaminated Produce

Methods used to isolate, detect, and identify Shi-gella spp. from foods can be divided into three major categories: conventional bacteriological, nucleic acid based, and biosensors. Presently, there is not one defi nitive method that has the robustness, rapidity, and effi cacy to be effective in isolating Shigella from foods. Clinical samples, which refl ect a much smaller number of matrices than foods, pose a challenge to laboratory personnel isolating Shigella (e.g., from feces). Currently, no enrichment medium exists to selectively grow Shigella from either food or fecal samples. This severely hampers the ability of laborato-ries to isolate Shigella from foods, particularly when this pathogen competes with the indigenous fl ora pres-ent in foods. Most methods pres-entail growth in broth medium which is followed by plating on selective agars. In the Bacteriological Analytical Manual (Andrews and Jacobsen, 2001; www.cfsan.fda.

gov/~ebam/bam-6.html), the authors detail one method to isolate shigellae from foods. Health Canada (http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/

microbio/index_e.html) uses a very similar scheme, with slight modifi cations. A protocol from the Inter-national Organization of Standards (ISO 21567:2004, Microbiology of food and animal feeding stuffs—

Horizontal method for the detection of Shigella spp.) offers another method.

A range of selective agar media is recommended to plate cultures after growth overnight in broths.

Two or three different selective media should be used to increase the chance of recovering Shigella. Growth of Shigella on MacConkey agar, a low-selectivity medium, is used to screen for lactose-negative colo-nies (Shigella are lactose negative). Eosin methylene blue and Tergitol-7 agar are alternative low-selectivity agars. Desoxycholate and xylose-lysine-desoxycholate agars are intermediate selective media and are pre-ferred media to isolate Shigella spp. Although most Shigella spp. do not ferment xylose, some species, e.g., S. boydii, have variable reactions and may be missed. Highly selective media include Salmonella-Shigella and Hektoen agars. Some Salmonella-Shigella spp., such

PCR. Several manufacturers provide kits, and most, if not all, require enrichment in broth. This added step may be suffi cient for some pathogens, but shigel-lae pose a more diffi cult task. Injured, weak, viable but nonculturable cells may persist in the population and may not be resuscitated by enrichment. There-fore, false-negative reactions may not be truly indica-tive of contamination. One alternaindica-tive to enrichment uses a fi lter that lyses the bacterial cell and sequesters the DNA in the membrane and can be used directly as a template in the reaction (Lampel et al., 2000). Real-time PCR has the advantage of reducing the Real-time for analysis. There are several different platforms that are available, each with their own strengths. In each system, the amplifi cation of PCR products can be monitored in real time, and in some cases, an extra step, such as using a probe within the reaction, can increase the specifi city of the reaction in a very short period of time. Also, much of this type of analysis is automated, meaning less chance of mistakes and reduced labor. As with conventional PCR, the same problems exist; templates in suffi cient quantity and free of PCR inhibitors prepared from all types of food matrices are critical. Most of these protocols require enrichment in broth for 6 to 18 h.

Another aspect of identifying causative agents of food-borne outbreaks of diarrheal disease is the ability to link the people affected in one region to other geographical locales and ultimately to the source of contamination. In this regard, pulse-fi eld gel electrophoresis surpasses most other nucleic acid-based typing methods. The CDC has established sev-eral surveillance programs to monitor food-borne outbreaks in the United States. These include PulseNet (http://www.cdc.gov/pulsenet/) and Food-Net (http://www.cdc.gov/foodnet/); others can be found at http://www.cdc.gov/ and, specifi cally for Shigella, at http://www.cdc.gov/ncidod/dbmd/phlis data/shigella.htm. The World Health Organization (WHO; http://www.who.int/emc/) also monitors out-breaks and publishes some of their fi ndings in the Weekly Epidemiological Record (http://www.who.

int/wer/index.html). Ribotyping, another means of subtyping microbial pathogens that uses gel electro-phoresis patterns of PCR-amplifi ed RNA genes, sub-sequently digested with a specifi c endonuclease, has been used to establish a database to type and identify Shigella strains (Coimbra et al., 2001).

Other technologies

An alternative to agarose gel electrophoresis is the application of an enzyme-linked immunosorbent assay at the conclusion of a PCR run to detect the ipaH gene of Shigella spp. from stool specimens (Set-Serological testing using polyvalent antiserum is

used to identify the Shigella groups A to D. A note of caution should be taken. EIEC causes the same disease, bacillary dysentery, as do the shigellae. Some O-antigen structures of EIEC strains share homology with O-antigen structures of some Shigella serotypes (Tulloch et al., 1973). Several serotypes of S. dysenteriae, S. fl exneri, and S. boydii have reciprocal cross-reactivity with E.

coli O antigens of the Alkalescens-Dispar bioserogroup or EIEC.

Although S. sonnei has only one serotype, two forms of this pathogen exist on agar plates. Form (or phase) I is virulent and has a smooth colony texture, whereas form II is irreversibly avirulent and has a rough colony phenotype on agar surfaces. Both forms carry distinct antigens; therefore, antiserum targeting both forms should be applied to identify immuno-logically S. sonnei isolates.

DNA-based assays

Several types of DNA-based assays have been applied to detect Shigella spp. in foods. Initially, DNA probes and PCR were developed to detect Shi-gella from clinical samples. PCR, an in vitro ampli-fi cation system, is a much more sensitive means of detecting the presence of microbial pathogens than conventional bacteriology and the use of DNA probes. As for using a PCR-based assay to detect Shigella in foods, primers are selected that target one specifi c virulence gene, ipaH (Wang et al., 1997).

The advantage of these primers is that the ipaH gene is present in multiple copies in the virulence plasmid (fi ve copies) and chromosome (seven genes/homo-logues; cognates) of Shigella (Jin et al., 2002;

Wei et al., 2003). Hence, the 5 to 12 target sequences increase the sensitivity of the assay com-pared to primers targeting just one copy of a gene.

Also, in instances when the large virulence plasmid is lost, these particular primers do target copies of the ipaH gene in the chromosome, ensuring an opportunity for a successful reaction. Unlike other methods that may take several days to draw a conclusion regarding the presence of Shigella in foods, PCR assays can yield a result in less than 1 day. One potential problem for PCR-based assays is the presence of inhibitors of the reaction deriving from the food matrix. To ensure that negative results are truly that, control reactions using washes or homogenates with target DNA added, are essential for accuracy. Therefore, template preparation is one of the key steps in ensuring the correct result in ana-lyzing foods by PCR.

Two approaches are being entertained, namely, whether or not to include an enrichment step prior to

laboratories. Foods present a different scenario. Food matrices have diverse effects on the ability of the pathogen to either grow or survive. In addition, the matrices pose a diffi cult challenge to the food analyst attempting to isolate and even detect the presence of Shigella in foods. An effective means of isolating and detecting the presence of Shigella in foods remains elusive. However, recent advances in instrument tech-nology are now at the stage of assessing these instru-ments for analysis of different environmental matrices.

Technology of today can be the basis of instruments in the near future that will result in analysis being completed in real time and being automated and por-table, two assets that will defi nitely impact food safety and food defense.

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CONCLUDING REMARKS

Diseases caused by the four species of Shigella remain a global health issue, whether in developed or developing countries. Epidemic and pandemic out-breaks are attributed to these pathogens and are par-ticularly acute in human environments that are crowded, have poor sanitation, and present an appro-priate milieu for facilitating rapid transmission of shigellae via the fecal-oral route. Children under the age of 5 years often have the highest mortality rate in developing countries, usually for the aforementioned conditions. The emergence of multiple antibiotic resistance strains poses another formidable challenge to health care workers treating bacillary dysentery.

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