Yersinia enterocolitica and Yersinia pseudotuberculosis
FOOD PROCESSING OPERATIONS THAT INFLUENCE THE NUMBER, SPREAD, OR
CHARACTERISTICS OF ENTEROPATHOGENIC
YERSINIA
Slaughter pigs often carry pathogenic Y. entero-colitica and Y. pseudotuberculosis in the oral cavity, particularly in tonsils and feces (Fredriksson-Ahomaa and Korkeala, 2003b; Niskanen et al., 2002, 2008).
The presence of symptomless carriers together with
Table 7. Effect of temperature on activity of virulence-related genes in Y. enterocoliticab
Genes/function
Effect of temp on gene expressiona
26°C 37°C
inv/invasion ↑ ↓
irp/iron uptake ↑ ↓
rfb/lipopolysaccharide ↑ ↓
yst/enterotoxin production ↑ ↓
virF/transcriptional activator ↓ ↑
yadA/adhesion to animal cells, serum resistance
↓ ↑
ysc, yop/secretion of Yops ↓ ↑
ail/attachment, serum resistance
↓ ↑
a↑, upregulation; ↓, downregulation.
bModifi ed from the work of Straley and Perry (1995).
be a problem (Alexandrino et al., 2004). Thus, investigations have been undertaken to develop rapid and reliable methods, such as PCR, for detecting pathogenic Yersinia, especially Y. enterocolitica, in food samples.
Detection of Y. enterocolitica and Y. pseudotuberculosis Using PCR
Several methods have been developed to detect Y. enterocolitica in food and environmental samples using PCR (Fredriksson-Ahomaa and Korkeala, 2003a). Many of these methods use primers targeted to the virF or yadA gene located on pYV (Table 8).
Because of possible plasmid loss during culturing, PCR methods targeted to chromosomal virulence genes have also been established. The ail gene located in the chromosome of pathogenic Y. enterocolitica strains is the most frequently used target. Some of the PCR methods have been created to detect more than one gene at the same time. The method most fre-quently used to detect PCR products has been elec-trophoresis in an agarose gel; however, the use of real-time monitoring by fl uorescence techniques has substantially increased. There are already some real-time PCR methods designed for qualitative detection of pathogenic Y. enterocolitica in food samples (Table 8). The Yersinia methods are based on dual-labeled probes (Jourdan et al., 2000; Boyapalle et al., 2001; Fredriksson-Ahomaa et al., 2007a) or SYBR green dye (Fukushima et al., 2003). There are, so far, two standardized conventional PCR methods for detection of pathogenic Y. enterocolitica in foods.
Method A is based on a one-step PCR with primers targeted to the ail gene in the chromosome, while
method B uses a two-step PCR with primers targeted to yadA on the pYV. These two methods have been published by the Nordic Committee on Food Analy-sis (NCFA, 1998).
Numerous commercial DNA purifi cation kits are available, and some of these kits have also been used in PCR assays designed for Y. enterocolitica. DNA extraction procedures using silica particles or chelex resin have commonly been used, as they are rapid and simple to use; however, they are not necessarily the most effective methods to remove inhibitors from complex matrices (Kaneko et al., 1995; Sandery et al., 1996; Jourdan et al., 2000; Boyapalle et al., 2001;
Vishnubhatla et al., 2001; Fredriksson-Ahomaa et al., 2007a). Buoyant density centrifugation, with some variations, has been used to separate Y. enterocolitica from food samples to remove inhibitors and to pre-vent false-positive results due to DNA originating from dead cells (Thisted Lambertz et al., 2000; Wolffs et al., 2004; Fukushima et al., 2007). Knutsson et al.
(2002) have designed a selective enrichment medium, Yersinia PCR-compatible enrichment medium, for Y. enterocolitica, which can be used prior to PCR to increase the sensitivity and decrease the risk of false-positive results due to detection of dead cells.
Detection of Y. enterocolitica and Y. pseudotuberculosis Using Culture Methods
It is generally easier to fi nd Y. enterocolitica and Y. pseudotuberculosis in clinical specimens of infected individuals than to fi nd them in asymptomatic carriers or in foods and environmental samples. During acute gastroenteritis or with organ abscesses, pathogenic Yersinia organisms are often dominant bacteria and
Table 8. Detection of Yersinia enterocolitica and Yersinia pseudotuberculosis in food samples using PCR Gene region(s) or gene(s) Type of PCR Detectiona Y. enterocolitica Y. pseudotuberculosis Reference(s) Plasmid
virF Nested AGE NCFA (1998)
yadA Nested AGE NCFA (1998)
Single Real time Wolffs et al. (2005)
Chromosome
ail Semi-nested AGE Sandery et al. (1996)
Nested AGE NCFA (1998)
Single Real time Jourdan et al. (2000),
Fredriksson-Ahomaa et al. (2007a)
yst Single AGE Özbas et al. (2000)
Single Real time Vishnubhatla et al. (2001)
Plasmid and chromosome
virF, ail, inv Multiplex AGE Kaneko et al. (1995)
virF, ail Multiplex AGE Nilsson et al. (1998)
yadA, 16S rRNA Multiplex AGE Lantz et al. (1999)
yadA, ail Multiplex AGE Boyapalle et al. (2001)
aAGE, agarose gel electrophoresis; Real time, real-time monitoring.
can easily be isolated by direct plating on conventional enteric media. Because of the high number of other bacteria and the low number of pathogenic strains of Yersinia in food and environmental samples, direct isolation, even on selective media, is seldom success -ful (Fredriksson-Ahomaa and Korkeala, 2003a). To increase the number of Yersinia strains in these sam-ples, enrichment in liquid media prior to isolation on solid media is required. Several different methods are available for isolation of Y. enterocolitica from food and environmental samples (Table 9). Far less infor-mation is available on reliable methods for recovery of Y. pseudotuberculosis from foods. The most widely used selective enrichment broth is irgasan-ticarcillin-potassium chlorate broth, which has been shown to be effi cient for recovery of strains of bioserotype 4/O:3. Niskanen et al. (2002) have shown that cold enrichment for 2 weeks in phosphate-buffered saline broth, supplemented with 1% mannitol and 0.15%
bile salts, is productive for isolation of Y. pseudotu-berculosis, especially followed by alkali treatment.
Alkali treatment with 0.25 to 0.50% KOH for 20 s has widely been used for isolation of Yersinia in foods (FDA, 2001; ISO, 2003; NCFA, 2003).
Many different selective plating media devel-oped for enteropathogens have been used for isola-tion of Yersinia (Fredriksson-Ahomaa and Korkeala, 2003a). Of the traditional enteric media, the most widely used is MacConkey (MAC) agar. Several entirely new media for isolation of Yersinia have been designed to improve selectivity. Most of the media have been designed for Y. enterocolitica; how-ever, Y. pseudotuberculosis is usually also able to grow on them, but its growth is mostly poor and delayed. Cefsulodin-irgasan-novobiocin (CIN) agar, according to Schiemann (1979), and Salmonella-Shigella deoxycholate calcium chloride agar, accord-ing to Wauters et al. (1988), are widely used for bioserotype 4/O:3 due to their high selectivity and commercial availability (ISO, 2003; NCFA, 2003).
However, differentiation of Yersinia from competing organisms, such as Aeromonas, Citrobacter, Enter-obacter, Klebsiella, Morganella, Proteus, and Serra-tia, can be diffi cult. The CIN agar is most commonly used for both Y. enterocolitica and Y. pseudotubercu-losis. MAC agar has been used alongside CIN and is generally considered to be useful for the isolation of Y. pseudotuberculosis, despite its low selectivity.
Y. enterocolitica and Y. pseudotuberculosis can be identifi ed with commercial rapid identifi cation kits, like API 20E, API Rapid 32 IDE, and Micronaute E.
The API 20E system, widely used for identifi cation of presumptive Yersinia isolates, has been shown to be accurate in identifying Y. enterocolitica and Y. pseudo-tuberculosis. This kit has high positive identifi cation rates of 93% and 90% for Y. enterocolitica and Y. pseudotuberculosis, respectively, when incubated at a temperature between 25°C and 30°C instead of 37°C for 18 to 24 h (Neubauer et al., 1998).
The majority of Y. enterocolitica isolates recov-ered from nonhuman sources are considrecov-ered non-pathogenic; thus, it is important to assess the pathogenicity of isolates (Fredriksson-Ahomaa and Korkeala, 2003a). This can be done rapidly and with high specifi city with DNA-based methods. Several PCR and DNA colony hybridization assays have been designed to verify the pathogenicity of Y. enteroco-litica isolates. The methods are based on specifi c seg-ments of the virulence plasmid or the chromosomal DNA with known virulence functions. Currently, two oligonucleotide probes are available from the FDA’s (U.S. Food and Drug Administration) Center for Food Safety and Applied Nutrition for identifi cation of pathogenic Y. enterocolitica (FDA, 2001). These probes are specifi c for the ail gene in the chromosome and the virF on the pYV. In addition, microarray chip technology has shown increased importance and potential in rapidly determining a complete gene pro-fi le in pathogenic microorganisms. Myers et al. (2006) have recently developed a microarray chip combined
Table 9. Isolation methods of Yersinia enterocolitica and Yersinia pseudotuberculosis most commonly used for food samplesa Enrichment medium
(conditions) Isolation medium/media (conditions) Y. enterocolitica Y. pseudotuberculosis Reference Not selective
TSB (22–25°C, 1 day) CIN (32°C, 18 h); MAC (22–25°C, 48 h) APHA (1992)
PBS (4°C, 14–21 days) CIN (32°C, 18 h); MAC (22–25°C, 48 h) APHA (1992)
Slightly selective
PSB (10°C, 10 days) CIN and MAC (30°C, 1 day) FDA (2001)
PSB (25°C, 1–3 days) CIN (30°C, 24 h) ISO (2003)
Selective
ITC (25°C, 2 days) SSDC (30°C, 24 h) ISO (2003)
MRB (22–25°C, 2–3 days) CIN (32°C, 18 h) APHA (1992)
a PBS, phosphate-buffered saline; PSB, phosphate-buffered saline broth with sorbitol and bile salts; ITC, irgasan-ticarcillin-potassium chlorate broth; SSDC, Salmonella-Shigella-sodium deoxycholate-calcium chloride agar plate; MRB, modified Rappaport broth; TSB, tripticase soy broth.
The RAPD assay, which is simple and quick to perform but may possess a low reproducibility, has been used in several studies to characterize Y. enterocolitica strains (Fredriksson-Ahomaa et al., 2006a). It allows discrimination between strains belonging to different bioserotypes and also, in some cases, between strains belonging to the same bioserotype. RAPD is also able to distinguish Y. pseudotuberculosis strains at the subserotype level; thus, it has been used in two out-break studies by Makino et al. (1994) and Kageyama et al. (2002).
CONCLUDING REMARKS
Yersiniosis is an infectious disease caused by Yersinia, food-borne yersiniosis being due to Yersinia enterocolitica or Yersinia pseudotuberculosis. Most human illness worldwide is caused by Y. enterocolitica.
The reported cases of yersiniosis are mainly sporadic and outbreaks are uncommon. However, outbreaks of Y. pseudotuberculosis infection, often in school-children, have occurred in Finland and Japan. The primary transmission route of human yersiniosis is proposed to be fecal-oral via contaminated food.
Some epidemiological studies have linked Y. entero-colitica infection with consumption of uncooked or undercooked pork. In the United States, household preparation of chitterlings (traditional dish made of intestines of pigs in the southern United States) has often been associated with Y. enterocolitica O:3 infec-tions. Pathogenic Y. enterocolitica has frequently been recovered only from slaughter pigs. By remov-ing the head together with tonsils and tongue prior to evisceration and by using a mechanized bung cutter in connection with enclosing the anus and rectum in a plastic bag, the contamination of pathogenic Yersinia could probably be reduced. Despite the fact that Y. enterocolitica and Y. pseudotuberculosis are impor-tant food-borne pathogens, these bacteria have sel-dom been isolated from foods. The low isolation rate with multiplex PCR for detection and characterization
of four virulence genes in Y. enterocolitica. Multiplex PCR was used to amplify the virF, ail, and yst genes.
Using this technique, important virulence genes of Y. enterocolitica could simultaneously be detected directly from pasteurized whole milk.
Molecular Characterization
In epidemiological studies, differentiation of Y. enterocolitica and Y. pseudotuberculosis into sub-types is necessary to identify contamination sources and routes. Molecular techniques represent valuable alternatives for subtyping of Yersinia. Several DNA-based methods have been used in molecular typing of Y. enterocolitica and Y. pseudotuberculosis (Table 10).
Pulsed-fi eld gel electrophoresis (PFGE) has proved to be highly discriminatory for molecular typing of Yersinia; thus, this method is a commonly used tech-nique in epidemiological studies of Y. enterocolitica and Y. pseudotuberculosis. PFGE allows subtyping of Yersinia strains belonging to the same serotype (Niskanen et al., 2002; Fredriksson-Ahomaa et al., 2006a). Amplifi ed fragment length polymorphism, a recently adopted PCR-based typing method, also allows differentiation of Y. enterocolitica strains within serotype-related clusters (Fearnley et al., 2005).
Both techniques have been used to investigate the relationship between genotypes and host sources (Fredriksson-Ahomaa et al., 2006b; Kuehni-Boghenbor et al., 2006). Ribotyping, random amplifi cation of polymorphic DNA (RAPD) assay, and restriction endonuclease analysis of the plasmid (REAP) have also frequently been applied to characterize entero-pathogenic Yersinia strains (Table 10). Ribotyping has been shown to be a useful tool for molecular typ-ing, especially to characterize Y. pseudotuberculosis strains (Voskressenskaya et al., 2005). Fukushima et al. (1994, 1998) have demonstrated differences in the geographical distribution among Y. pseudotuberculo-sis strains belonging to the same serotype using REAP.
Table 10. Frequently used methods for molecular subtyping of Yersinia enterocolitica and Yersinia pseudotuberculosis Typing methoda Y. enterocolitica Y. pseudotuberculosis Reference(s)
AFLP Fearnley et al. (2005), Kuehni-Boghenbor et al. (2006)
RAPD Fredriksson-Ahomaa et al. (2006a)
Makino et al. (1994), Kageyama et al. (2002)
REAP Fredriksson-Ahomaa et al. (2006a)
Fukushima et al. (1994, 1998), Han et al. (2003)
Ribotyping Fredriksson-Ahomaa et al. (2006a)
Voskressenskaya et al. (2005)
PFGE Favier et al. (2005), Fredriksson-Ahomaa et al. (1999, 2006a, 2006b,
2007b), Falcao et al. (2006), Lambertz et al. (2007)
Niskanen et al. (2002, 2003, 2008), Nuorti et al. (2004), Jalava et al.
(2004, 2006)
aAFLP, amplifi ed fragment length polymorphism.
is probably due to the low sensitivity of the culture methods available. Using PCR, pathogenic Y. entero-colitica has been detected with a high frequency in raw pork and pork products. The host range of Y. pseudotuberculosis is broad, but the principal res-ervoir hosts are believed to be rodents, wild birds, and domestic animals (especially pigs and ruminants).
In the reported outbreaks of Y. pseudotuberculosis, fresh produce and untreated surface water have been implicated in the illness. A combination of direct con-tact with wildlife feces during the storage and cross-contamination of the equipment are the most likely contributing factors. Although several studies on the epidemiology of enteropathogenic Yersinia have been conducted, a lot of questions remain to be solved using DNA-based detection and characterization methods in the future.
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