Public Health Aspects of Foodborne Diseases Related to Swine
Foodborne diseases have high societal costs worldwide. According to the economic research service of the U.S. Department of Agriculture (USDA), the cost of foodborne Salmonellosis in the U.S. is estimated to be $2.4 billion per year. An estimated more than 13 million illnesses and 1800 deaths per annum are also attributed to foodborne illnesses caused by mainly bacterial pathogens (www.ers.usda.gov). Pork accounts for 25% of domestic meat consumption and associated products have been implicated as origins of foodborne outbreaks in the nation and globally (Molbak et al., 1999; Pontello et al., 1998; McGuire et al., 1993). The U.S. pork industry has been a net exporter since 1995. In addition, with increasing total meat production every year (84 billion pounds projected for this year alone with 1% increase from 2001), the demand for high quality pork, free of pathogens, is also increasing. Campylobacter spp and non-typhoidal Salmonella serovars are two of the leading foodborne pathogens. In relation to swine production, Salmonella remains the most important partly since public health important serotypes and multi-drug resistant strains are common in swine. As for Campylobacteriosis, little is known about its significance in the swine production but clearly poultry products, which are known reservoirs of the most common species, C. jejuni, are important. Swine are known to be reservoirs of the less common species, C. coli, which is responsible for less than 20% of human campylobacteriosis (OBrien et al., 2002). The USDA FSIS baseline survey implicated market hog as the second leading product with high prevalence of Campylobacter spp. (FSIS, 1996) implying the need for further understanding.
Previous studies of swine Salmonellosis in North Carolina revealed a pig prevalence of up to 48% (Funk et al., 2000). Our group also conducted a risk factor analysis and antimicrobial resistance. The findings on the former study indicate biosecurity measures (number of personnel per farm) as one of the significant factors associated with increased Salmonella shedding. Detailed results of the study are presented by J. Funk in this proceeding (Funk et al., 2001). In a cross-sectional study of antimicrobial resistance, we have also reported that antimicrobial resistance among Salmonella serovars is common. Particularly, the most common and public health important serovar Typhimurium (including var. Copenhagen) were found to be commonly multi-drug resistant (MDR) with two distinct penta-resistance types (R-types): Ampicillin, Chloramphenicol Streptomycin, Sulfamethoxazole and Tetracycline (ACSSuT) and Ampicillin, Kanamycin, Streptomycin, Sulfamethoxazole and Tetracycline (AKSSuT). Predominantly, the former type was found to be DT104 (Gebreyes et al., 2000).
The objectives of this study were:
- Determine secular trends of MDR types in swine.
- Determine antimicrobial susceptibility among Salmonella isolates from humans.
- Conduct preliminary phenotypic and genotypic comparison of human and swine isolates.
- Identify and characterize important genetic determinants.
- Enable us to discern a potential explanation why distinct MDR strain are common in swine but do not commonly cause disease outbreaks in human.
Origin of Isolates
Salmonella isolates from three different sources were included in this study. The first is field (non-clinical) samples from a longitudinal study of Salmonellosis in North Carolina (Funk et al., 2001). For this study, 484 S. Typhimurium (including var. Copenhagen) were included. The second was diagnostic human isolates from North Carolina Public Health Laboratory Services. So far, 215 Typhimurium isolates have been derived from counties with high concentration of swine farms. In addition, we included 13 (of total 39) swine veterinary diagnostic samples from the NC state Veterinary Diagnostic laboratory to determine genetic diversity.
Antimicrobial susceptibility to 12 antimicrobial agents was determined as recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Two methods were primarily used to determine antimicrobial susceptibility: Vitek Jr. (Biomerieux) and Kirby-Bauer disk diffusion methods. We tested susceptibility to ampicillin (A), chloramphenicol (C), streptomycin (S), sulfamethoxazole (Su), tetracycline (T), amoxillin/clavulanic acid (Ax), cephalothin (Cf), ceftriaxone (Cro), ciprofloxacin (Cip), kanamycin (K), amikacin (An) and gentamicin (Gm). Isolates with intermediate resistance to ceftriaxone were subjected to E-test for final confirmation. Quality control strains were tested routinely as recommended by NCCLS.
We determined genotypic similarity of isolates from different sources by pulsed field gel electrophoresis (PFGE) method using the protocol recommended by the CDC (Gautom, 1997). Analysis of genotypes was done by constructing dendrograms using Bionumerics (Applied Maths, Belgium) software. Dendrograms were constructed using the Unweighted Pair Group Method using Arithmetic means (UPGMA).
Identification of genetic determinants
We used Polymerase Chain Reaction (PCR) to identify genetic determinants with relevance in this study. These include genes encoding resistance to ? – lactams, such as ampicillin (blaPSE1, blaTEM), third generation cephalosporins (blaCMY-2), tetracycline resistance alleles (tetA, B, C, D, and G) and class-I integrons (int1). In addition, we tested for the carriage of Salmonella plasmid virulence genes (spvA, B, C, D and a regulator gene spvR) to explain specific findings in phenotypic and genotypic characterization.
During the three-year study, there was a significant decline of ACSSuT resistance type among S. Typhimurium (including var. Copenhagen) whereas a significant increase in the AKSSuT resistance type was observed (p < 0.05). This shift of strains has been seen among S. Typhimurium var. Copenhagen isolates while Typhimurium isolates consistently showed the latter resistance type. Similar observations were noted by the National Antimicrobial Resistance Monitoring System (NARMS) study since 1997 (NARMS, 1997). There has not been any recorded change in management and ecologic factors during this study period. This shift of MDR strains is explained by molecular characterization of genetic determinants as described in the subsequent section.
Analysis of antimicrobial susceptibility among Typhimurium isolates derived from human diagnostic specimens revealed that more than 80% of the isolates were susceptible to all antimicrobial agents tested (Figure 1). On the other hand, more than 90% serovar Typhimurium (including Copenhagen) isolates from swine exhibited an MDR pattern, predominantly AKSSuT R-type. None of the MDR types among human diagnostic specimens exhibited AKSSuT resistance type but 20 isolates (out of 33 MDR) in Typhimurium from humans and 11 of 15 of swine exhibited ACSSuT R-type. Even though epidemiological relationship between the swine and human isolates could not be discerned based on this study, as the samples were conveniently collected from different sources, we believe valuable data could be deduced as a preliminary finding for the antimicrobial resistance and genetic diversity of isolates.
Another interesting observation among human isolates includes the occurrence of ceftriaxone resistance in 2 of the isolates tested so far. In contrast, we have not found third generation cephalosporin resistance among isolates from swine (non-clinical and diagnostic) [n=2190]. Ceftriaxone resistance has been exhibited by two serovars: Typhimurium and Heidelberg. Further genetic characterization of this resistance revealed that these isolates carry blaCMY-2 gene (Figure 2). This gene, to our knowledge, has not been identified from serovar Heidelberg, which is one of the serovar commonly found both in humans and swine.
MDR Typhimurium isolates from the three different sources including swine nonclinical, swine diagnostic and human diagnostic samples were genotyped using PFGE (Figure 3). As clustering of isolates shows and supported by the phenotypic findings, diagnostic MDR isolates clustered distinctly different from those derived from swine for research (non-clinical) purposes. This finding together with the phenotypic findings at the national level (NARMS, 1997) could have important implication that MDR isolates that are commonly present in swine (AKSSuT) may not be the major part of foodborne problems in humans as well as the common swine disease problems though these MDR types are found more commonly in the swine gastrointestinal tract.
To characterize genetic differences between distinct pentaresistant isolates derived from different sources, we performed PCR analysis of representative isolates for the presence of Salmonella plasmid virulence gene, spvR. We found that most of the isolates from diagnostic sources carry spvR (10 of 13) but only 1 of 6 isolates from non-clinical carried this gene. The presence of this gene is highly correlated with the R-type. None of the isolates with kanamycin resistance (though diagnostic) carried this gene.
In the longitudinal study of swine salmonellosis, we have been interested in the secular trend of frequency of MDR types. Among var. Copenhagen, there was a clear shift of MDR type from ACSSuT, which were predominant at the beginning of the study, to AKSSuT at the end of the study (p<0.05). Similar MDR types were found to predominate among animal isolates tested by the NARMS (NARMS, 1997). Isolates with this R-type were very rare among diagnostic samples. The human arm of NARMS, which evaluates diagnostic human Salmonella samples also found consistently low frequency of this R-type in comparison to the clinically more common ACSSuT R-type. According to the NARMS human report of 2000, among the total of 303 Typhimurium isolates tested, 27.7% exhibited ACSSuT R-type, whereas only 9.2% exhibited AKSSuT R-type. This difference could be attributed to the difference in carriage of important virulence factors by different strains such as Salmonella plasmid virulence (spv).
Salmonella plasmid virulence genes are known to be common among serovars including Typhimurium, Dublin, Enteritidis, Choleraesuis and other pathogenic serovars. The spv region contains virulence gene clusters. Absence or mutations in this region is reported to attenuate or inactivate the ability of Salmonella to proliferate in intestinal tissues. The 90 kilo base pairs virulence plasmid of serovar Typhimurium (also known as pSLT) is required for full virulence after oral infection (Gulig and Curtiss, 1987). The absence of spv genes among AKSSuT isolates in this study could explain two important questions. First, it answers why only certain R-types may be commonly identified from diagnostic specimens but not the other (AKSSuT) and the second is why isolates with AKSSuT R-type are commonly identified from healthy (non-clinical) isolates. The absence of spv genes may make the strains incapable of causing clinical diseases since spv is an important virulence factor and at the same time the lack of this factor may make strains better fit to survive in the gastrointestinal tract of pigs. In general, the findings presented here together with the observations at the national level may imply that not all Salmonella serovar Typhimurium are equally pathogenic and their zoonotic potential also differs accordingly suggesting the need for understanding further beyond the serotyping level in addressing issues of foodborne illnesses.
Frequency of antimicrobial resistance among human diagnostic isolates revealed that unlike the common resistance to particularly tetracycline and ? -lactams among swine isolates, human isolates were predominantly susceptible to all antimicrobials tested (Figure 1). As this is a preliminary study where samples were collected based on convenience, the finding could be partly explained by sampling bias. The national average reported in 2000 indicated that 49.5% (ranging between 35 and 77% per state) of Typhimurium are pan-susceptible (NARMS, 2000), which is lower than our findings (80%). This could be due to several factors including the lower number of antimicrobials that we tested and the lower sample size that we used. We are currently proposing to study persistence of antimicrobial resistance in the swine production environment to study the role of human and the environment in dissemination of antimicrobial resistance and its interface with swine production.
As a preliminary investigation of genotypic diversity of Typhimurium isolates from different sources including swine diagnostic, non-clinical and human diagnostic samples, we conducted PFGE fingerprinting. As shown in Figure 3, we detected diversity of isolates collected from swine in non-clinical specimens from those of diagnostic ones. The diagnostic isolates from humans and swine were closely related. Therefore, the clonal identity of isolates was dichotomized based on the type of isolate (clinical versus non-clinical) but not the host (human versus swine). This is also supported by R-type and genetic identity of virulence factors among diagnostic specimens from both swine and humans (Figure 3). These findings in general support the hypothesis that not all MDR Typhimurium has similar capability of causing disease and only distinct clonal strains are able to do so. This is also supported by recent reports in other organisms linking between virulence and ecological abundance (Day et al 2001).
We have characterized antimicrobial resistance genes among Typhimurium isolates from swine and humans. We characterized the genes among the common AKSSuT R-type and the findings are in the process of publication (Gebreyes et al., 2002). What really is of interest among the resistance phenotypes and resistance genes from human isolates is the presence of ceftriaxone resistance encoded by blaCMY-2 gene (Figure 2). This gene encodes resistance to third generation cephalosporins. From the public health point of view, this finding is of high significance since ceftriaxone is the sole resort in treating children with resistant infections. We detected ceftriaxone resistance only among the human isolates (n=215). None of the swine isolates tested so far were found to be resistant to third generation cephalosporins (n=2170). The two human isolates with ceftriaxone resistance were of two different serovars: Typhimurium and Heidelberg. In the past few years, this phenotype and resistance gene has been reported from few serovars mainly Typhimurium and Newport. The presence of this gene among Heidelberg poses additional risk to pork production since serovar Heidelberg is among the top five serovars both in human and swine. Therefore, from the pork industry point of view, there is clear need of limiting such resistant strain from accessing swine farms. Strict biosecurity measures play a significant role in achieving this goal. Further characterization of this strain and other resistant ones important to the swine industry and with relevance to public health are underway.
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Dr. Wondwossen Gebreyes
Dr. Gebreyes is Assistant Professor of Molecular Epidemiology, Department of Farm Animal Health Resources Management at the College of Veterinary Medicine at North Carolina State University in Raleigh, NC. In 1990, he completed his doctor of Veterinary Medicine at Addis Ababa University, Debrezeit, Ethiopia and in 2001 completed his Ph.D. at North Carolina State University . His thesis advisor was Crag Altier and his thesis title was Molecular Epidemiology of Multi-drug Resistant Salmonella in Swine.