Molecular analysis of environmental and human isolates of Salmonella typhi.

Molecular characterization of a total of 54 isolates of Salmonella typhi from Santiago, Chile, was performed by pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases: XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Thirteen of the 54 isolates were obtained from environmental sources (sewage and river water), and the rest were isolates from clinical cases of typhoid fever. Considerable genetic diversity was detected among the human isolates obtained in 1994, as evidenced by the presence of 14 to 19 different PFGE patterns among 20 human isolates, with F (coefficient of similarity) values ranging from 0.69 to 1.0 (XbaI), 0.61 to 1.0 (AvrII), and 0.70 to 1.0 (SpeI). A total of eight phage types were detected among these 20 isolates, with 50% possessing the E1 or 46 phage type. There was no correlation between PFGE pattern and phage types. Similar diversity was seen among 21 isolates obtained in 1983, with 17 to 19 PFGE patterns detected and F values of 0.56 to 1.0 (XbaI), 0.55 to 1.0 (AvrII), and 0.67 to 1.0 (SpeI). Comparison of these two groups of human isolates obtained 11 years apart indicated that certain molecular types of S. typhi are shared and are able to persist for considerable periods. A similar degree of genetic diversity was also detected among the environmental isolates of S. typhi, for which 10 to 12 different PFGE patterns were detected among the 13 isolates analyzed, with F values ranging from 0.56 to 1.0 (XbaI), 0.52 to 1.0 (AvrII), and 0.69 to 1.0 (SpeI). Certain molecular types present among the environmental isolates of S. typhi were also found among the human isolates from the same time period, providing evidence for the epidemiological link between environmental reservoirs and human infection.     

Analysis of Salmonella enteritidis strains isolated from food-poisoning cases in Taiwan by pulsed field gel electrophoresis, plasmid profile and phage typing

AIMS: To establish the molecular typing data for Salmonella enteritidis due to its increasing role in Salmonella infections in Taiwan. METHODS AND RESULTS: Sixty-three Salm. enteritidis strains isolated from related and unrelated patients suffering from food-borne poisoning during 1991-97 were collected and subjected to pulsed field gel electrophoresis (PFGE), plasmid analysis and phage typing. For PFGE, XbaI, SpeI and NotI restriction enzymes were used for chromosomal DNA digestion. The results showed that, for these 63 Salmonella strains, 10 PFGE pattern combinations were found. Of these, pattern X3 S3 N3 was the major subtype, since 46 strains isolated from different locations at different times during 1991-97 showed this PFGE pattern. Plasmid analysis showed only three plasmid profiles and phage typing showed that most of the Salmonella strains were of the phage type PT4. CONCLUSION: Most of the Salm. enteritidis strains circulating in Taiwan are of very similar genetic types or are highly related and that strains of PFGE pattern X3 S3 N3 are the prevalent and recirculating strains of Salm. enteritidis which caused food-poisoning cases in Taiwan in 1991-97. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information that in Salmonella infection, certain subtypes of Salm. enteritidis should be scrutinized.     

Application of Random Amplified Polymorphic DNA analysis to differentiate strains of Salmonella typhi and other Salmonella species

A Random Amplified Polymorphic DNA (RAPD) fingerprinting method was developed to differentiate isolates of Salmonella serotype typhi ( S. typhi ) and other Salmonella isolates. A panel of five primers was used to examine 63 isolates of Salm. typhi , including 56 strains isolated in Taiwan and seven strains obtained abroad. Twenty-one RAPD types were revealed using the RAPD fingerprinting method. An RAPD with primer 6032 yielded a polymorphism in a 350 bp fragment that differentiated the attenuated vaccine strain Salm. typhi Ty21a from the rest of the Salm. typhi strains. Strains of Salm. typhi were divided into five types with primer D14307. Primer D14307 also proved capable of discrimination among 65 other Salmonella isolates representing 42 different serotypes. The bacterial DNA used in this RAPD protocol was obtained using a commercially available DNA extraction kit (GeneReleaser). The DNA of various strains of Salmonella from this simple extraction procedure could be discriminated within a few hours using the RAPD technique.     

Pulsed-field gel electrophoresis, plasmid profiles and phage types for the human isolates of Salmonella enterica serovar Enteritidis obtained over 13 years in Taiwan

AIMS: Plasmid profile, phage typing, and pulsed-field gel electrophoresis (PFGE) patterns of 124 Salmonella Enteritidis strains isolated in 1998-2002 in Taiwan were analysed and the results were compared with those of the 63 strains obtained in 1991-1997, so that molecular subtypes and epidemic strains for Salmonella Enteritidis over a 13-year period (1991-2002) could be elucidated. METHODS AND RESULTS: A total of 124 strains of Salmonella Enteritidis isolated from human in Taiwan between 1998 and 2002 were analysed by PFGE, plasmid analysis and phage typing. The results obtained were compared with those of the 63 strains obtained in 1991-1997, so that the clonal relationships for a total of 187 strains obtained over 13 years could be elucidated. For PFGE, restriction enzymes XbaI, SpeI and NotI were used for chromosomal DNA digestion. Results showed 28 PFGE pattern combinations for the 187 Salmonella strains. Of them, pattern X3S3N3 was the major subtype as 130 strains isolated from different locations during 1991-2002 showed this PFGE pattern. For all these 187 strains, the genetic similarity was higher than 80%. Plasmid analysis showed 17 distinct types, which consist of one to four plasmids and the predominant phage type of those strains was PT4 (71.6%) and PT6a (13.4%). The three methods identified different degrees of polymorphism in the following order: plasmid profile (18 types, D = 0.659) > PFGE (28 types, D = 0.512) > phage typing (13 types, D = 0.438). As PFGE patterns, phage type and plasmid profile were combined for subtyping, the 187 strains could be grouped into 46 subtypes and the discriminatory index was raised to 0.795. For these 46 subtypes, the predominant one was X3S3N3/P1/PT4, which contained 77 (41%) isolates. CONCLUSIONS: Most of the Salmonella Enteritidis strains from sporadic cases were with pattern X3S3N3. They were the prevalent and may be the epidemic strains found in Taiwan during 1991-2002. The present study suggested that the several variants were derived from a single clonal line and the genome for strains of Salmonella Enteritidis are highly conserved over a 13-year period (1991-2002). SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here are useful for epidemiolgical study of salmonellosis caused by Salmonella Enteritidis in Taiwan. Comparing the data of the present study with those obtained for strains from other countries, the major subtypes for Salmonella Enteritidis infection in the world can be elucidated.     

Pulsed-field gel electrophoretic identification of Salmonella enterica serovar Typhimurium live vaccine strain Zoosaloral H

Salmonella enterica subsp. enterica serovar Typhimurium ( Salm. Typhimurium) live vaccine strain Zoosaloral H was characterized by pulsed-field gel electrophoresis (PFGE). Each of the two suitable restriction enzymes, Xba I and Spe I, produced a unique restriction fragment pattern for this live vaccine strain which was not shared by field isolates of the same serovar. The characteristic fragment pattern proved to be stable during a 22 month observation period and was also not altered after animal passage of the vaccine strains. Thus PFGE analysis proved to be a helpful tool in the identification of Salm. Typhimurium live vaccine strain Zoosaloral H and its differentiation from wild-type isolates of the same serovar.     

121株甲型副伤寒病原菌的噬菌体型和脉冲场凝胶电泳型

目的认识甲型副伤寒疫病区甲型副伤寒沙门菌的噬菌体型和脉冲场凝胶电泳（PFGE）型,确定噬菌体型和PFGE型之间的关系以及菌型的分布和流行率。方法采用沙门菌组合噬菌体和SpeI、XbaI消化染色体DNA的PFGE对来自玉溪市7县（区）的121株甲型副伤寒菌进行分型。结果121株菌存在4个完全噬菌体型或1个噬菌体型;用SpeI或XbaI消化产物分别得出以SpeI01、SpeI02或XbaI01占优势的5种或4种PFGE型,SpeI01型和SpeI02型分别占37．2％和57．9％,XbaI01型占95．1％。结论121株菌的噬菌体型与PFGE型之间无一致性联系,PFGE型的SpeI01和SpeI02或XbaI01是玉溪地区的主要流行型,采用SpeI和XbaI的PFGE是鉴别甲型副伤寒菌流行克隆的一项有用技术。     

Pulsed-field gel electrophoresis as an epidemiological tool for clonal identification of Aeromonas hydrophila

Pulsedfield gel electrophoresis (PFGE) was used to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections that occurred over approximately 1 month in a French hospital. Five isolates from patients and 10 isolates from the water supply were characterized by biotyping and antibiotic susceptibility patterns and compared with 10 epidemiologically unrelated strains isolated from patients and rivers, by PFGE of digests of chromosomal DNA. Five environmental and four clinical isolates belonged to the same biotype and antibiotic susceptibility pattern type. The endonucleases XbaI, SpeI and SwaI gave satisfactory profiles whereas DraI did not. The profiles were stable, reproducible and discriminatory. The 10 epidemiologically unrelated strains exhibited 10 different patterns after digestion with XbaI , the least expensive, suitable endonuclease. PFGE is a rapid and discriminatory technique for the typing of Aeromonas hydrophila where a common origin of infection is suspected.     

Genetic diversity of Salmonella enterica serovar Paratyphi A from different geographical regions in Asia

AIMS: Subtyping of Salmonella Paratyphi A isolates from India, Pakistan, Indonesia and Malaysia was carried out by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these isolates from different endemic countries. METHODS AND RESULTS: A total of 39 human isolates of Salmonella Paratyphi A from Pakistan, India, Indonesia and Malaysia were studied using PFGE analysis following digestion of chromosomal DNA with XbaI. Seven isolates from Pakistan were resistant to ampicillin, tetracycline and cotrimoxazole. It was noted that Salmonella Paratyphi A isolates obtained from outbreaks in India had limited genetic diversity and probably belonged to closely related clones. Significant genetic homogeneity was observed among antimicrobial-resistant isolates from Pakistan and antimicrobial-sensitive isolates from Pakistan and Indonesia, respectively. CONCLUSIONS: PFGE was a useful subtyping technique to differentiate Salmonella Paratyphi A from different endemic countries. However, it fails to differentiate the antimicrobial-resistant and -sensitive strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study verify the usefulness of PFGE in characterizing and comparing strains of Salmonella Paratyphi A. Our study suggests that a limited number of clones are responsible for paratyphoid fever in these countries.     

The presence of major world-wide clones for phage type 4 and 8 Salmonella enterica serovar Enteritidis and the evaluation of their virulence levels by invasiveness assays in vitro and in vivo

Seventy-seven animal isolates of Salmonella enterica serovar Enteritidis (S. Enteritidis) obtained from the United States were analyzed by phage typing and pulsed field gel electrophoresis (PFGE). Thirty-nine strains were found with phage types (PT) 4, 8, and 13a. When the chromosomal DNA of these 39 isolated strains with PT4, 8, and 13a were digested with XbaI, SpeI and NotI, followed by PFGE analysis, 28 strains were found with a pattern combination of X4S4N4, which was the major subtype. When PFGE patterns of the US isolates with PT 4 and 8 were compared with those of the Taiwanese and German isolates, pattern X3S3N3 was confirmed to be the world-wide subtype shared by PT 4 isolates, as previously reported, while pattern X4S4N4 was newly found to be the most common subtype shared by PT 8 strains. The presence of such major world-wide clones, however, does not necessarily mean that these clones are highly virulent, at least not according to the results of invasiveness assays using cultured human intestinal epithelium cell line Int-407 and living BALB/mice.     

DNA fingerprinting of human isolates of Salmonella enterica serotype Paratyphi B in Malaysia

AIMS: DNA fingerprinting of Salmonella enterica serotype Paratyphi B isolated in Malaysia during 1982-83, 1992 and 1996-2002 was carried out by pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility tests and D-tartrate utilization tests to assess the extent of genetic diversity of these isolates in Malaysia. METHODS AND RESULTS: Eighty-six human isolates and one food isolate of Salm. Paratyphi B were analysed by PFGE, antimicrobial susceptibility tests and D-tartrate utilization tests. Sixty-five strains were D-tartrate-negative (dT-) while 22 strains were D-tartrate-positive (dT+). Thirty-seven per cent of the Salm. Paratyphi B strains were resistant to one or more antimicrobial agents. PFGE analysis clearly distinguished the dT- and dT+ strains into two clusters based on the unweighted pair group average method (UPGMA). Twenty-two XbaI-pulsotypes were observed among the 65 dT- strains while 17 XbaI-pulsotypes were observed among the 22 isolates of Salm. Paratyphi B dT+. CONCLUSIONS: The present study showed that PFGE was very discriminative with 33.7% of the strains yielding distinct fingerprints. Paratyphoid fever in Malaysia is probably caused by one predominant, endemic clone of Salm. Paratyphi B dT- with various subtypes. There was no association between the pulsotypes and the severity of the disease indicating that the severity of the disease is probably multifactorial. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the present study verify the usefulness of PFGE in characterizing strains of Salm. Paratyphi B. This is the first report on the application of PFGE on a large collection of Salm. Paratyphi B in Malaysia.     

Improvement of strain discrimination by combination of RAPD with PFGE for the analysis of the swine isolates of Salmonella enterica serovar Choleraesuis

Salmonella enterica serovar Choleraesuis (S. Choleraesuis) can cause salmonellosis in pigs and humans. Currently, the most common method used for the subtyping of this Salmonella serovar is pulsed field gel electrophoresis (PFGE) using XbaI as a DNA digestion enzyme. In this study, we compared and combined PFGE with the randomly amplified polymorphic DNA method, for the typing of 95 S. Choloraesuis strains isolated from diseased pigs. Using PFGE with XbaI, with AvrII, and with SpeI digested DNA, 29, 74, and 40 patterns, respectively, were obtained. Also, 53, 15, and 35 strains, respectively, belong to the major patterns X1, A1, and S1. When these three digestion patterns were combined, 83 PFGE pattern combinations were obtained. On the other hand, using RAPD with selected primer alone generated 76 patterns, and 11 strains which fell within a single X1A1S1 PFGE combination pattern were discriminated into 10 patterns. Thus, for S. Choloraesuis, PFGE with AvrII allowed higher discrimination than PFGE with XbaI, and some of the PFGE groupings obtained by combining the XbaI, AvrII and SpeI digestion patterns were further subdivided by the RAPD method.     

Molecular epidemiological analysis of bloodstream isolates of Candida albicans from a university hospital over a five-year period

We assessed the genetic relations and epidemiological links among bloodstream isolates of Candida albicans, which were obtained from a university hospital over a period of five years. The 54 bloodstream isolates from the 38 patients yielded 14 different karyotypes, 29 different patterns after digestion with SfiI (REAG-S), and 31 different patterns after digestion with BssHII (REAG-B) when analyzed using three different pulsed-field gel electrophoresis (PFGE) typing methods. In 11 patients with serial bloodstream isolates, all strains from each patient had the same PFGE pattern. The dendrograms for all of the strains revealed that the distribution of similarity values ranged from 0.70 to 1.0 in the REAG-S patterns, and from 0.35 to 1.0 in the REAG-B patterns. Overall, the combination of the three different PFGE methods identified 31 distinct types, reflecting the results obtained using the REAG-B alone different. different Five PFGE types were shared among 22 isolates from 12 patients. These types of strains were more frequently associated with central venous catheter-related fungemia than the other 26 type strains (92% versus 31%; P < 0.005). Of five PFGE types, four isolates were determined to be epidemiologically related: each of these types was primarily from two or three patients who had been hospitalized concurrently within the same intensive care unit. Our results suggest that the REAG-B constitutes perhaps the most useful PFGE method for investigating C. albicans candidemia and also shows that a relatively high proportion of C. albicans candidemia may be associated with exogenous acquisition of clonal strains.     

Characterization of Salmonella enterica serovar Heidelberg from Turkey-Associated Sources

Salmonella enterica serovar Heidelberg strains are frequently associated with food-borne illness, with recent isolates showing higher rates of resistance to multiple antimicrobial agents. One hundred eighty S. enterica serovar Heidelberg isolates, collected from turkey-associated production and processing sources, were tested for antimicrobial susceptibility and compared by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis. The potential for the transfer of resistance between strains was studied by conjugation experiments. PFGE analysis using XbaI digestion identified eight clusters (based on 90% similarity), with the largest containing 71% of the isolates. Forty-two percent of the isolates were resistant to at least 1 of the 15 antimicrobial agents tested, and 4% of the isolates were resistant to 8 or more antimicrobial agents. Resistances to streptomycin (32%), tetracycline (30%), and kanamycin (24%) were most commonly detected. Interestingly, the XbaI PFGE profiles of selective multidrug-resistant strains (n = 22) of S. enterica serovar Heidelberg from turkey-associated sources were indistinguishable from the predominant profile (JF6X01.0022) detected in isolates associated with human infections. These isolates were further differentiated into seven distinct profiles following digestion with the BlnI enzyme, with the largest cluster comprising 15 isolates from veterinary diagnostic and turkey processing environments. Conjugation experiments indicated that resistance to multiple antimicrobial agents was transferable among strains with diverse PFGE profiles.     

Detection of Salmonella typhi by polymerase chain reaction

A rapid and sensitive method for detection of Salmonella typhi would help in preventing the spread of outbreaks and in clinical diagnosis. In order to develop unique PCR primers to detect Salm. typhi , ribosomal RNA genes from Salm. typhi (Rawlings) were cloned in pUC18. The resulting clone was confirmed by sequencing. The cloned DNA fragment contained the 5S, part of the 23S rRNA genes and the 5S-23S spacer region (EMBL/GenBank accession No. U04734).
It was expected that the 5S-23S spacer region is divergent unlike the highly conserved 23S+5S genes. This was confirmed by comparison with the rRNA gene sequences in the EMBL/GenBank database. A pair of PCR primers specific for Salm. typhi was obtained, based on this spacer region sequence. The specificity of this pair of primers was tested with 54 Salm. typhi strains (of 27 different phage types). All these Salm. typhi strains showed the positive 300 bp PCR product with this pair of primers. Six other Salmonella species as well as six other non- Salmonella bacteria were tested and none showed the 300 bp PCR product. The sensitivity of the detection level was 0·1 pg of pure Salm. typhi genomic DNA, or approximately 40 Salm. typhi cells in a spiked food sample. This pair of primers therefore has the potential for development into a diagnostic tool for the rapid diagnosis of typhoid fever.     

Characterization of Erwinia amylovora strains from Bulgaria by pulsed-field gel electrophoresis

The aim of this study was to characterize genetically Bulgarian Erwinia amylovora strains using pulsed-field gel electrophoresis (PFGE) analysis. Fifty E. amylovora strains isolated from different hosts, locations, as well as in different years were analysed by PFGE after XbaI, SpeI, and XhoI digestion of the genomic DNA. The strains were distributed into four groups according to their XbaI-generated profile. About 82% of the strains displayed a PFGE profile identical to that of type Pt2. Three strains belonged to the Central Europe Pt1 type. Two new PFGE profiles, not reported so far, were established--one for a strain isolated from Malus domestica and another for all Fragaria spp. strains. The same grouping of the strains was obtained after analysis of the SpeI digestion patterns. On the basis of PFGE profiles, after XbaI and SpeI digestion, a genetic differentiation between the strains associated with subfamily Maloideae and subfamily Rosoideae was revealed. The presence of more than one PFGE profile in the population of E. amylovora in Bulgaria suggests a multiple source of inoculum.     

Investigation of the genetic diversity among isolates of Salmonella enterica serovar Dublin from animals and humans from England,Wales and Ireland

AIMS: To assess the degree of genetic diversity among animal Salmonella Dublin UK isolates, and to compare it with the genetic diversity found among human isolates from the same time period. METHODS AND RESULTS: One hundred isolates (50 human and 50 animal) were typed using plasmid profiling, XbaI-pulsed field gel electrophoresis (PFGE) and PstI-SphI ribotyping. Antimicrobial resistance data to 16 antibiotics was presented, and the presence of class-I integrons was investigated by real-time PCR. Seven different plasmid profiles, 19 ribotypes and 21 PFGE types were detected. A combination of the three methods allowed clear differentiation of 43 clones or strains. Eighteen isolates were resistant to at least one antimicrobial; five of them were multi-resistant and of these, only three presented class I integrons. CONCLUSIONS: Ribotyping data suggest the existence of at least three very different clonal lines; the same distribution in well-defined groups was not evident from the PFGE data. The existence of a variety of clones in both animals and humans has been demonstrated. A few prevalent clones seem to be widely disseminated among different animal species and show a diverse geographical and temporal distribution. The same clones were found in animals and humans, which may infer that both farm and pet animals may act as potential vehicles of infection for humans. Some other clones seem to be less widely distributed. Clustering analysis of genomic fingerprints of Salmonella Dublin and Salm. Enteritidis isolates confirms the existence of a close phylogenetic relationship between both serotypes. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes the utility of a multiple genetic typing approach for Salm. Dublin. It gives useful information on clonal diversity among human and animal isolates.     

Comparison of type A enterotoxigenic Staphylococcus aureus strains isolated from geographically far distant locations by pulsed field gel electrophoresis

Staphylococcal enterotoxin A (SEA) is one of the major staphylococcal enterotoxins which may cause food-borne outbreaks. In order to investigate the difference in genomic types and to elucidate the most disseminated strains for enterotoxin A-producing strains of Staphylococcus aureus , a total of 60 SEA Staph. aureus strains isolated from food and clinical samples in Taiwan and 30 strains of the same enterotoxigenic type of strains obtained from geographically far distant locations were compared for their pulsed field gel electrophoresis (PFGE) patterns. The rare cutting endonuclease Sma I generated 10 distinct genome patterns for the 60 local SEA isolates and 15 and eight genome patterns, respectively, for the 20 and 10 SEA strains originally isolated from the USA and other countries. The local isolates are less diverse in genome patterns as compared to the US isolates. Of all these PFGE patterns, a certain pattern, such as pattern 3, is shared by the food and clinical isolates and the local and foreign isolates. Thus, although SEA Staph. aureus strains from geographically far distant locations showed considerable genetic diversity, PFGE pattern 3 strain might be one of the most disseminated strains.     

Phage types, plasmid profiles and chromosomal restriction profiles of Salmonella enterica subsp. enterica ser. enteritidis (S. enteritidis) isolated in Poland in 1999-2000]

Salmonella Enteritidis strains are the most often isolated Salmonella serovars in Poland. In the present study, phage typing, plasmid profile analysis, and PFGE have been applied to characterize 140 Polish S. Enteritidis isolates originated from human cases of salmonellosis and from other sources. The typing phages of Ward and colleagues scheme were used to type a total of 140 S. Enteritidis strains coming from Poland. All 140 strains were typable and six different phage types were observed. A total of 125 (89%) of 140 isolates examined belonged to PT 4. The others PTs were represented by small amount of strains (PT1-2, PT6-6, PT7-1, PT8-4 and PT21-2 strains). Among all tested isolates six different plasmid profiles were observed. Of the 140 examined strains, 128 (91.4%) contained the 57 kb plasmid alone. After XbaI digestion four distinct pulse field chromosomal restriction profiles among studied S. Enteritidis were observed. XbaI and SpeI chromosomal restriction profiles of S. Enteritidis PT4 were identical with reference strain profiles. Our findings confirmed earlier suggestions that the increase of human salmonellosis cases in Poland was caused by S. Enteritidis PT4 and was due to consumption of contaminated food. This study confirmed the importance of using PFGE in combination with phage typing, plasmid typing and antibiotic resistance testing for studying the epidemiology of S. Enteritidis.     

Antimicrobial susceptibility and pulsed-field gel electrophoresis of Shigella sonnei strains in Malaysia (1997-2000)

AIMS: Antimicrobial resistance of Shigella sonnei from Malaysia was determined and subtyping was carried by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these strains. METHODS AND RESULTS: A total of 62 isolates of S. sonnei from sporadic cases of shigellosis in different parts of Malaysia were studied by antimicrobial susceptibility test and PFGE. Approximately 35.5% of the strains showed resistance to three or more antimicrobial agents. Eight resistant phenotypes, i.e. RI to RVIII, was defined. Resistant phenotype RV and RVIII only appeared in year 2000. PFGE analysis with NotI and XbaI restriction showed that a great heterogeneity existed at the DNA level among Malaysian S. sonnei isolates. Fifty-eight NotI and 61 XbaI-PFGE profiles were observed in 63 S. sonnei isolates, including ATCC 11060 isolate. Drug sensitive isolates displayed very different profiles from drug-resistant isolates, with a few exceptions. Isolates of resistant phenotype RVI (SXTr.TETr.STRr) showed a greater similarity among each other compared with isolates of resistant phenotype RI and drug-sensitive isolates. CONCLUSION: Multi-drug-resistant S. sonnei were circulated in different parts of Malaysia and the emergence of new resistant phenotype was observed. Wide genetic variations among Malaysian S. sonnei were observed and the drug-sensitive strains could be differentiated from drug-resistant strains by PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study verifies the usefulness of PFGE in characterizing and comparing strains of S. sonnei. Minor variations among S. sonnei isolates could be detected by PFGE.     

Genotypic characterization of Salmonella typhi by amplified fragment length polymorphism fingerprinting provides increased discrimination as compared to pulsed-field gel electrophoresis and ribotyping

Amplified fragment length polymorphism (AFLP) is a recently developed, PCR-based high resolution fingerprinting method that is able to generate complex banding patterns which can be used to delineate intraspecific genetic relationships among bacteria. In the present study, AFLP was evaluated for its usefulness in the molecular typing of Salmonella typhi in comparison to ribotyping and pulsed-field gel electrophoresis (PFGE). Six S. typhi isolates from diverse geographic areas (Malaysia, Indonesia, India, Chile, Papua New Guinea and Switzerland) gave unique, heterogeneous profiles when typed by AFLP, a result which was consistent with ribotyping and PFGE analysis. In a further study of selected S. typhi isolates from Papua New Guinea which caused fatal and non-fatal disease previously shown to be clonally related by PFGE, AFLP discriminated between these isolates but did not indicate a linkage between genotype with virulence. We conclude that AFLP (discriminatory index=0.88) has a higher discriminatory power for strain differentiation among S. typhi than ribotyping (DI=0.63) and PFGE (DI=0.74).