Single-enzyme Amplified Fragment Length Polymorphism Research Articles

Vagococcus sp. a porcine pathogen: molecular and phenotypic characterization of strains isolated from diseased pigs in Brazil

Vagococcus spp. is known for its importance as a systemic and zoonotic bacterial pathogen even though it is not often reported in pigs. This is related to the pathogen misidentification due to the lack of usage of more discriminatory diagnostic techniques. Here we present the first report of Vagococcus lutrae in swine and the characterization of Vagococcus fluvialis and Vagococcus lutrae isolated from diseased animals. Between 2012 and 2017, 11 strains with morphological characteristics similar to Streptococcus spp. were isolated from pigs presenting different clinical signs. Bacterial identification was performed by matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry and confirmed by 16S rRNA sequencing and biochemical profile. Strains were further genotyped by single-enzyme amplified fragment length polymorphism (SE-AFLP). Broth microdilution was used to determine the minimal inhibitory concentration of the antimicrobials of veterinary interest. Ten strains were identified as V. fluvialis and one was identified as V. lutrae. The SE-AFLP analysis enabled the species differentiation with specific clustering of all V. fluvialis separately from the V. lutrae strain. Most strains presented growth in the maximum antibiotic concentration values tested for eight of the 10 analyzed antimicrobial classes. The observed resistance pattern can represent a problem for veterinary and producers in the treatment of diseases associated Vagococcus spp. in swine production. Vagococcus species may also be a risk for pig industry workers. The data described here will be of great value in further understanding the behavior of this pathogen in animal production.

Phenotypic, molecular and genomic characterization of Actinobaculum suis isolated from swine in Brazil

Urinary tract infections (UTI) are considered one of the most important diseases of sows due to its close relationship with reproductive problems such as reduced litter size, increase in the rate of return to estrous, vulvar discharge, abortion, mastitis and anestrus. Actinobaculum suis is one of the main agents involved in porcine urinary tract infection and is responsible for the most severe and fatal cases in sows. In the present report, 23 A. suis strains isolated from a sow and boars in Brazil were identified by PCR and further characterized by broth microdilution, molecular typing by pulsed-field gel electrophoresis (PFGE), single-enzyme amplified fragment length polymorphism (SE-AFLP), and whole-genome sequencing. All strains were sensitive to ceftiofur, linezolid, nitrofurantoin, quinupristin-dalfopristin and vancomycin. Ciprofloxacin, daptomycin, lincomycin, erythromycin and tylosin resistance was observed in 100% of tested strains. Tetracycline and tigecycline also presented high resistance rates (87% and 30.4%, respectively). PFGE with eight different restriction enzymes and three programs did not enable strain characterization; however, all strains were typed by SE-AFLP that clustered strains according to their origin, thus proving an effective tool for A. suis genotyping. Whole-genome sequencing and comparative analysis enabled species differentiation from closely related genus. This is the first report of genomic characterization of A. suis.

Use of a modified AFLP protocol to discriminate Salmonella enterica subsp.enterica serovar Enteritidis isolates

Salmonella enterica subsp. enterica ( S .) serovar Enteritidis is one of the main pathogens involved in food-borne diseases worldwide. In epidemiological investigations of food-related salmonellosis, subtyping is necessary to improve preventive and control measures. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis is a modified AFLP that uses only one restriction enzyme to produce DNA fragments that are selectively amplified by PCR. In order to assess the applicability of SE-AFLP in S. Enteritidis typing, one hundred and eight strains isolated from poultry, swine and also from human salmonellosis outbreaks in Southern Brazil were analyzed. Strains from other countries and six different S. enterica serovars were also included as controls. SE-AFLP was able to distinguish S . Enteritidis from the other S. enterica serovars analyzed. However, most of S . Enteritidis strains isolated from poultry, salmonellosis outbreaks and most of the strains from other countries shared the same predominant pattern. The low genetic diversity identified in S . Enteritidis suggests that the strains analyzed are clonally related and one predominant SE-AFLP genotype is widely spread in Southern Brazil.

Isolation and molecular characterization of Arcobacter butzleri and Arcobacter cryaerophilus from the pork production chain in Brazil

ABSTRACT: Arcobacter is an emerging zoonotic pathogen, and the major transmission routes to humans are the handling or consumption of contaminated raw/undercooked food products of animal origin, water and seafood. The isolation and identification of Arcobacter species are not routine in clinical laboratories; therefore, its true incidence in human infections may be underestimated. The present study aimed to isolate and characterize Arcobacter from carcasses and fecal samples collected at swine slaughterhouses and from meat markets in São Paulo State, Brazil. The isolates were identified using multiplex-PCR to differentiate the species and analyzed by single-enzyme amplified fragment length polymorphism (SE-AFLP). Arcobacter spp. were isolated from 73.0% of swine carcasses, 4% of fecal samples and 10% of pork samples. A. butzleri was the most prevalent species identified, followed by A. cryaerophilus. Interestingly, the carcasses presented higher frequency of A. butzleri isolation, whereas only A. cryaerophilus was isolated from fecal samples. SE-AFLP enabled the characterization of A. butzleri and A. cryaerophilus into 51 and 63 profiles, respectively. The great genetic heterogeneity observed for both species corroborates previous reports. This study confirms the necessity for a standard isolation protocol and the improvement of molecular tools to further elucidate Arcobacter epidemiology.

Genotypic Characterization of Arcobacter spp. Isolated from Chicken Meat in Brazil.

Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii are Gram-negative pathogenic microorganisms that cause watery diarrhea and septicemia in humans. The aims of this study were to detect the presence of Arcobacter spp. in chicken meat from butcher shops in São Paulo (Brazil) and to verify their virulence genes and genotypic profiles. A total of 300 chicken cuts were analyzed. The results show the presence of Arcobacter spp. in 18.3% of samples, which were identified as A. butzleri (63.6%) and A. cryaerophilus (36.3%). All strains were positive for the virulence genes ciaB and mviN, followed by cj1349 (98%), pldA (94.4%), cadF (72.7%), tlyA (92.7%), hecA (49%), irgA (47.2%), and hecB (34.5%). These strains were subjected to single-enzyme amplified fragment length polymorphism. Nineteen genotypic profiles were obtained for A. butzleri, and 17 for A. cryaerophilus. These results confirm the presence of virulent strains of A. butzleri and A. cryaerophilus in the chicken meat in Brazil. The presence of potentially virulent strains of Arcobacter highlights a possible public health risk, particularly with respect to ingestion of undercooked foods and cross-contamination from uncooked foods during food preparation and contaminated utensils.

Profiling of Leptospira interrogans, L. santarosai, L. meyeri and L. borgpetersenii by SE-AFLP, PFGE and susceptibility testing—a continuous attempt at species and serovar differentiation

Leptospirosis is a widespread systemic zoonosis, considered as reemerging in certain developing countries. Although the cross agglutinin absorption test is still considered the standard method for Leptospira identification, it presents several disadvantages. The aim of this study was to characterize Leptospira spp. isolated from various hosts by genotyping and broth microdilution susceptibility testing in an attempt to differentiate Leptospira species, serogroups and serovars. Forty-seven isolates were studied. They were previously serotyped, and species confirmation was performed by 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) and pulsed-field gel electrophoresis (PFGE) analysis enabled the distinction of L. interrogans from L. santarosai, L. meyeri and L. borgpetersenii in two main clusters. Among L. interrogans, it was possible to differentiate into two new clusters the serogroup Icterohaemorrhagiae from the serogroups Canicola and Pomona. L. santarosai isolates presented higher genetic variation than the other species in both techniques. Interestingly, the minimum inhibitory concentration (MIC) cluster analysis also provided Leptospira serogroup differentiation. Further studies are necessary regarding serovar Bananal isolates, as they presented the highest MIC values for most of the antimicrobials tested. All studied techniques successfully distinguished Leptospira species and serogroups. Despite being library-dependent methods, these approaches are less labor intensive and more economically viable, particularly SE-AFLP, and can be implemented in most reference laboratories worldwide to enable faster Leptospira typing.

Characterization and amino acid metabolism performances of indigenous Oenococcus oeni isolated from Chinese wines

Oenococcus oeni is a multiple physical stress-tolerant lactic acid bacterium that plays an important role in wine making. It is often added as a starter culture to carry out malolactic fermentation (MLF). In this study, a total of 22 out of 127 lactic acid bacteria, isolated from Chinese wines undergoing MLF, were identified as O. oeni by species-specific PCR and 16S rRNA sequencing. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis showed that all strains could be typed under these conditions, and three main groups were determined by cluster analysis, which showed intraspecific homology higher than 69 %. Eight strains, representative of SE-AFLP clusters, were tested for malolactic activity. Significant differences were observed among strains with regard to the amount of malic acid consumed. Seventeen amino acids in different wines that were inoculated by 4 O. oeni strains, respectively, were analyzed before and after MLF. The results indicated that the amino acid metabolism of the 4 strains was significantly different between each strain.

High level of genetic diversity among Steinernema monticolum in Korea revealed by single-enzyme amplified fragment length polymorphism

Steinernema monticolum was first described from a mountainous forest at sites of Sancheong, Gyeongnam province in Korea. Since S. monticolum is one of the most commonly isolated entomopathogenic nematodes from Korea, it is desirable to investigate the diversity of this species. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analyses were used to differentiate 32 S. monticolum populations. Our results revealed a high level of genetic diversity within S. monticolum at the population level. On the geographic scale, SE-AFLP analysis revealed that there was no correlation between the genetic similarity of populations of this species and their geographical proximity.

Molecular analysis of ciprofloxacin resistance and clonal relatedness of clinical Escherichia coli isolates from haematology patients receiving ciprofloxacin prophylaxis

Widespread use of fluoroquinolones has led to increased levels of resistance in clinical isolates of Escherichia coli. We investigated the evolution of ciprofloxacin susceptibility and molecular epidemiology of clinical E. coli isolates in haematology patients receiving ciprofloxacin prophylaxis on the population and individual patient level. From August 2006 through December 2007 we collected all E. coli isolates (n = 404) from surveillance and infection-site cultures from 169 haematology patients receiving ciprofloxacin prophylaxis. Analysis of the gyrase A (gyrA) gene was performed by denaturing gradient gel electrophoresis (DGGE) in 364 isolates and clonal relatedness was determined by the single-enzyme amplified fragment length polymorphism (seAFLP) technique in 162 isolates. One hundred of these isolates were also subjected to qnrA analysis. The average number of samples per patient was 2.4 (maximum 20) and 122 (30%) of 404 E. coli isolates were resistant to ciprofloxacin. In 124 patients only ciprofloxacin-susceptible strains were detected. DGGE revealed 11 different gyrA sequence patterns and, based on AFLP analysis, there was evidence of selection of ciprofloxacin-resistant strains under antibiotic pressure, as well as the occurrence of genetically indistinguishable ciprofloxacin-resistant and -susceptible E. coli isolates within one patient. Clonal dissemination of ciprofloxacin-resistant E. coli was observed, but did not predominate. The genetic evolution of clinical E. coli isolates in haematology patients receiving ciprofloxacin prophylaxis is characterized by selection of ciprofloxacin-resistant strains. However, we did find evidence for de novo resistance mutation in ciprofloxacin-susceptible E. coli in individual patients under selective pressure.

Genotypic characterization ofStaphylococcus aureusisolated from bovines, humans, and food in Indonesia

The present study determined the genetic relationships between 41 Staphyloccocus (S.) aureus isolates from bovines, humans, and food using a single enzyme amplified fragment length polymorphism (AFLP) technique. We evaluated the prevalence of staphylococcal enterotoxin (SE) genes and other virulence gene determinants by PCR. The identification of S. aureus was based on culturing and biochemical tests, and by amplifying a specific section of the 23S rRNA gene. PCR amplification of the SE genes (sea, seb, sec, see, seg, seh, and sei) singly or in combination was observed. Most isolates of bovine origin harbored hla (84%) and cap5 (74%), while most isolates from humans harbored hla (73%), cap8 (91%), and fnbA (100%). Strains from food sources were positive for hla (100%), cap5 (100%), and cap8 (64%) unlike isolates from humans or bovines. A single enzyme AFLP analysis revealed a correlation between AFLP clusters of some strains and the source of the isolates The genotypic results of the present study might help to better understand the distribution of prevalent S. aureus clones among humans, bovines, and food and will help control S. aureus infections in Indonesia.

Cluster of Cases of Salmonella enterica Serotype Rissen Infection in a General Hospital, Italy, 2007

In 2007, three strains of Salmonella enterica serotype Rissen (S. Rissen) were isolated in the laboratory of diagnostic microbiology of the General Hospital of Prato, Tuscany, Italy, over a 1 month and half interval of time. The first isolate was recovered on January 26 from an outpatient with enteritis. Then, two strains were isolated on February 16 and March 11 respectively, from central venous catheters of patients who were being hospitalized in two departments of the Hospital. An epidemiologically linked cluster of cases of salmonellosis was suspected. The three strains were submitted to single enzyme-amplified fragment length polymorphism (SE-AFLP) and XbaI macrorestriction and pulsed-field gel electrophoresis (PFGE) that yielded undistinguishable profiles. Epidemiological investigations failed to identify a common source of infection within the Hospital. Moreover, the third patient had been exclusively total parenteral nutrition fed since his admission with a stomach cancer diagnosis. The first patient had a community-acquired infection, but the source of her illness was uncertain. Twenty-five further isolates identified in the years 2004-2007 in the same geographical area showed distinctly different PFGE and SE-AFLP patterns. The three patients seemed to represent a cluster of epidemiologically unrelated cases caused by a previously never recognized S. Rissen strain. Rapid subtyping of isolates is essential in the early investigation of potential outbreaks, but synthesis of conventional and molecular epidemiological investigation and availability of surveillance data is often critical to prevent the initiation of time-consuming, expensive and ineffective further investigations and control interventions.

Characterization of Multiple Helicobacter bizzozeronii Isolates From a Finnish Patient With Severe Dyspeptic Symptoms and Chronic Active Gastritis

Helicobacter pylori is the primary cause of gastritis and peptic ulceration in humans. In a minority of patients with upper gastrointestinal symptoms, long tightly coiled spiral bacteria, provisionally named "Helicobacter heilmannii," are observed in gastric biopsies. These bacteria are extremely fastidious and only one previous study has succeeded in obtaining an isolate in vitro. We used two different selective media to isolate "H. heilmannii" from the gastric mucosa of a Finnish patient presenting with severe dyspeptic symptoms. The isolates were characterized by testing for urease and catalase activity, by using light and electron microscopy, and by sequencing of the partial 16S rRNA and ureAB genes. Single-enzyme amplified fragment length polymorphism (sAFLP) was used to analyze the genetic diversity among the isolates. We obtained 15 isolates from different gastric biopsies prior and three after unsuccessful treatment of the patient. The isolates were identified as Helicobacter bizzozeronii. Eradication therapy was unsuccessful most probably due to high level of resistance to metronidazole. Persistent colonization by the same H. bizzozeronii clone was confirmed by sAFLP, however, small differences between the profiles suggested long-term colonization of the patient. Helicobacter bizzozeronii remains the only "H. heilmannii" species isolated from human gastric mucosa although it has been an infrequent observation among "H. heilmannii"-infected patients in PCR-based screening studies. The relevance of H. bizzozeronii and other potentially zoonotic gastric Helicobacter spp. in human disease remains to be determined.

Molecular Epidemiology of Japanese Avian Pasteurella multocida Strains by the Single-Enzyme Amplified Fragment Length Polymorphism and Pulsed-Field Gel Electrophoresis

Molecular epidemiology analyses of the 36 clinical isolates of Pasteurella multocida from various avian hosts in Japan between 1976 to 2007 including 5 reference strains from the U.S.A., Taiwan and Indonesia were performed by employing the single-enzyme amplified fragment length polymorphism (SE-AFLP) comparison with the classical ApaI-based pulsed-field gel electrophoresis (PFGE). As the results, SE-AFLP gave 21 profiles while PFGE gave 20 profiles. The Simpson's index of diversity analysis indicated that SE-AFLP gave a high discrimination power than PFGE. This concluded that SE-AFLP is a higher discrimination power than PFGE to differentiate avian P. multocida isolates in Japan. In addition, the genetical profiles suggested that there is the evolution of somatic serotype 3 strain in the indigenous host of Japan.

Use of Random Amplified Polymorphic DNA Analysis and Single-Enzyme Amplified Fragment Length Polymorphism in Molecular Typing of Ornithobacterium Rhinotracheale Strains

Ornithobacterium rhinotracheale (ORT) is a bacterium common to commercial poultry and wild birds throughout the world. It is also known as a causative agent of respiratory diseases. A total of 93 ORT isolates originating from chickens, pigeons, ostriches, quail, turkeys, and an Asian crested goshawk (Accipiter trivirgatus) in Taiwan, between 2004 and 2006, were used in this study. High genetic similarity (97%-100%) in 16S rRNA sequence was revealed among the 50 randomly selected isolates, in addition to a reference strain (ATCC-51464) and seven reference sequences from GenBank. In order to obtain a greater genetic discrimination among the ORT isolates, random amplified polymorphic DNA (RAPD) and single-enzyme amplified fragment length polymorphism (SE-AFLP) methods were further conducted. The results showed that both RAPD and SE-AFLP assays showed higher discriminatory abilities than the 16S rRNA sequence assay. Genetic clustering revealed that chicken- and quail-origin isolates were genetically distinct from those of the ostrich, pigeon, and Asian crested goshawk-origin isolates. However, among the two typing methods, the turkey-origin isolates showed diverse genetic characteristics to domestic avian species. With this information, ecologic and epidemiologic studies could be furthered for the reduction and control of ORT transmission in Taiwan.

Enterobacter colonisation in newborn infants: predictors, follow-up and implications for infection control

Outbreaks with Enterobacter spp. have been described frequently in neonatal intensive care units (NICUs). This study investigated the factors that determine whether a neonate becomes colonised with Enterobacter spp., how long colonisation continues and whether the termination of isolation measures leads to spread of the organism. Neonates transferred from the NICUs of tertiary care hospitals were screened for the presence of Enterobacter spp. and any potential predictors for colonisation recorded. Those infected were monitored during their hospital stay and colonised neonates were screened every month for six months. Isolation infection control precautions were lifted and all neonates were screened for the presence of Enterobacter spp. six and 12 months later. Fifteen colonised neonates and 33 non-colonised controls were identified for study. Multivariate analysis showed that antibiotic therapy for more than three days and an Apgar score of <8 after 1 min were independently associated with Enterobacter spp. colonisation. Molecular typing using single-enzyme amplified-fragment length polymorphism (seAFLP) analysis revealed 22 different seAFLP genotypes. Three infants remained colonised with the same Enterobacter genotype after discharge; however, most neonates lost their strain or became colonised with another genotype. Lifting infection control measures for neonates colonised with Enterobacter spp. in a neonatal ward did not lead to increased incidence of colonisation and none of the infants became infected. Isolating neonates with susceptible Enterobacter spp. was not found to be necessary.

Reinterpreting a community outbreak of Salmonella entericaserotype Enteritidis in the light of molecular typing

BackgroundIn November 2005, a large outbreak due to Salmonella enterica serotype Enteritidis (S. Enteritidis) was observed within children who had eaten their meals at 53 school cafeterias in Florence and the surrounding area. A total of 154 isolates of S. Enteritidis were recovered from human cases between November 2005 and January 2006. All strains were assigned phage type 8 (PT8) and a common XbaI pulsotype.This paper reports the findings of a molecular epidemiological investigation performed on 124 strains of S. Enteritidis isolated in the years 2005 and 2006 in Florence and the surrounding area, including the epidemic isolates.MethodsOne hundred twenty-four human isolates of S. Enteritidis identified in the period January 2005 – December 2006 were submitted to molecular typing by single enzyme – amplified fragment length polymorphism (SE-AFLP).ResultsMolecular subtyping by SE-AFLP yielded five different profiles. In the pre-epidemic phase, type A included 78.4% of isolates, whereas only three (8.1%) belonged to type C. All isolates, but one, of the epidemic phase were indistinguishable and attributed to type C. In the post-epidemic period, a polymorphic pattern of SE-AFLP types was again recognized but type C accounted for 73.3% of the isolates during the first six months of 2006, whereas during the remaining six months type A regained the first place, including 52.0% of the isolates.ConclusionThe epidemic event was attributed to the emergence and clonal expansion of a strain of S. Enteritidis PT8-SE-AFLP type C. Circulation of the epidemic clone was much more extensive than the surveillance and traditional laboratory data demonstrated.

Use of single-enzyme amplified fragment length polymorphism for typing Clostridium perfringens isolated from diarrheic piglets

Clostridium perfringens is an important pathogen in human and veterinary medicine. In swine, the agent is responsible for necrotic enteritis and enterotoxemia characterized by diarrhea, weight loss, delayed development and, in some cases, death. In the present study amplified fragment length polymorphism analyses (AFLP) was used to characterize 54 C. perfringens strains isolated from swine presenting diarrhea. Analysis of the results showed 29 distinct profiles with discriminatory index equal to 0.97. Partial correlation between the origin of the isolates and groups was drawn, and correlation was possible in only 18.5% of the samples. Characterization of the strains in biotypes (A, B, C, D and E), production of beta-2 toxin and enterotoxin were performed by means of the polymerase chain reaction (PCR). Biotypes A, C and D were observed among the strains analyzed. All samples were positive for presence of the gene encoding beta-2 toxin and negative for the gene encoding enterotoxin. AFLP have shown to be a simple, fast, low cost method with high discriminative power and good reproducibility, presenting a great potential in epidemiological studies involving C. perfringens strains of animal origin.

Characterization of avian strains of Pasteurella multocida by restriction endonuclease and amplified fragment length polymorphism

Avian strains of Pasteurella multocida were typed by employing restriction endonuclease analysis (REA) and single enzyme-amplified fragment length polymorphism (AFLP) to evaluate their applicability for epidemiological studies of fowl cholera outbreaks. A total of 72 strains isolated from different avian species (chicken, duck, turkey, quail and goose) belonging to various geographical regions of India were characterized. REA using two different enzymes HhaI and HpaII produced 9 and 18 clusters respectively, whereas Single enzyme-AFLP recognized 32 patterns out of 72 strains typed. The study indicated that REA using HpaII is a simple and resource efficient method, however, further typing with more stringent and rapid method like Single enzyme-AFLP, could drastically enhance investigation in epidemiological studies of fowl cholera outbreaks.

Use of pulsed field gel electrophoresis (PFGE) and single-enzyme amplified fragment length polymorphism (SE-AFLP) to subtype isolates of Salmonella enterica serotype enteritidis

Serotype Enteritidis is still the main serotype infecting humans and poultry worldwide. Subtyping of isolates belonging to this serotype is difficult, because of the wide clonal circulation of a few bacterial clones. This study presents the results of the characterization of 49 isolates of S. Enteritidis identified at the southern Italy Centre for Enteric Pathogens (CEPIM) during the years 2002-2003 by the methods of Pulsed Field Gel Electrophoresis (PFGE) and Single-Enzyme Amplified Fragment Length Polymorphism (SE-AFLP). Clustering of the strains by SE-AFLP and PFGE is very similar, but the first technique is more rapid and user-friendly and does not require sophisticated equipment. Further work is needed for a more accurate assessment of SEAFLP, but preliminary results suggest it could be a promising support to epidemiological investigations.

Combination of pulsed-field gel electrophoresis (PFGE) and single-enzyme amplified fragment length polymorphism (SAFLP) for differentiation of multiresistant Salmonella enterica serotype typhimurium

To assess the combined application of plasmid profile typing, pulsed-field gel electrophoresis (PFGE) and PCR-based single-enzyme amplified fragment length polymorphism (SAFLP) for the differentiation of 18 multiresistant (MR) and one drug-sensitive strain of Salmonella enterica serotype typhimurium from humans and food animals. Strains were phage typed and tested for resistance to a panel of antimicrobial agents. Strains were also tested for the ability to transfer resistance either directly or by mobilization to standard strains of Escherichia coli K12. Plasmid DNA was extracted from both drug-resistant donor strains and from drug-resistant exconjugants. Total genomic DNA was characterized by PFGE following digestion with the restriction endonuclease XbaI. The resultant patterns were categorized and analyzed by dendrogram analysis using the Dice coefficient and by data clustering using unweighted pair-group arithmetic averaging (UPGMA). Isolates were also characterized and categorized by SAFLP. The levels of discrimination achieved by each method were assessed individually and in combination. Plasmid DNA was detected in all of the 18 MR isolates but, not in the drug-sensitive isolate. Using PFGE, 19 different profiles were identified, falling into eight major categories. However, by SAFLP, only eight profiles were observed. Subsequent investigations have demonstrated epidemiologic relationships within at least one of these SAFLP profile groupings. These studies have demonstrated that PFGE and SAFLP can be used independently for the differentiation of MR S. Typhimurium from humans and food animals. However, when used in combination, SAFLP can provide a format for broad epidemiologic groupings. These groupings can be further subdivided by PFGE to provide detailed information on putative strain relationships at the genotypic level.