Evaluation of DNA Fingerprinting by PFGE as an Epidemiologic Tool for Salmonella Infections

To evaluate DNA fingerprinting as an epidemiologic tool, pulsed-field gel electrophoresis (PFGE) was performed on isolates of Salmonella, including S. typhimurium, S. thompson, and S. enteritidis. Chromosomal DNA was digested with the restriction endonucleases Bln I and Xba I. The patterns of S. thompson and S. typhimurium isolates from various sources were different from one another. There was no correlation between the phage type and the digestion pattern of S. enteritidis isolates. Some strains belonging to one phage type were distinguished by their PFGE pattern in this study. These results suggest that the Bln I and Xba I digestion patterns of chromosomal DNA are useful for epidemiological analysis of an outbreak of Salmonella infection or food poisoning.     

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.     

Comparison of PCR-RFLP and PFGE for determining the clonality of Brucella isolates from human and livestock specimens

Brucellosis is an important zoonotic disease caused by different species of genus Brucella that are pathogenic for humans and a variety of animals. Accurate detection of Brucella spp. infection is important for control of disease. The aim of this study was to comparison of molecular genotyping of Brucella strains by Pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction -Restriction Fragment Length Polymorphism (PCR-RFLP) techniques.Twenty- seven Brucella spp. were isolated from human and animal samples. The isolates identified by conventional microbiological methods and confirmed using PCR for amplification of omp2a gene. Molecular typing of Brucella strains carried out by PCR-RFLP after PstI and PFGE of chromosomal DNA after XbaI enzyme digestion. The omp2a gene PCR Products with different patterns of PCR-RFLP were sequenced.The omp2a gene amplification of all human and animal Brucella isolates were positive for 1100 bp fragment. By PCR-RFLP analysis two genotypes/patterns for human isolates and four genotypes for animal isolates were obtained. In PFGE analysis totally, 7 common clones/clusters and 3 single clones were obtained.The results of this study showed the PFGE method is the more reliable and useful assay for molecular typing of Brucella strains and is more preferred to PCR-RFLP in determination of genetic similarity among human and animal Brucella isolates. The presented data showed PCR-RFLP analysis was not able to differentiate between B. melitensis biovars and vaccine strain.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.     

Pulsed-Field Gel Electrophoresis Is More Discriminating Than Multilocus Enzyme Electrophoresis and Random Amplified Polymorphic DNA Analysis for Typing Pyogenic Streptococci

The SmaI restriction endonuclease digestion patterns of chromosomal DNAs from 99 pyogenic streptococci belonging to Lancefield group A (41 Streptococcus pyogenes), group C (seven S. dysgalactiae, 11 \QS. equisimilis\W, three S. equi, eight S. zooepidemicus) and group G (25 human group G Streptococcus, four S. canis) were analyzed by pulsed-field gel electrophoresis (PFGE), and the results were compared with those previously obtained by multilocus enzyme electrophoresis (MLEE) and random amplified polymorphic DNA analysis (RAPD). PFGE revealed 93 distinct types among the 99 strains, and no patterns were common to strains of different species. The discriminatory power of PFGE was greater than that of MLEE and RAPD for groups A and G streptococci. The polymorphism among group C streptococci was similar with the three techniques. PFGE is, therefore, the most efficacious method for epidemiological typing of pyogenic streptococci. Received: 31 July 1996 / Accepted: 17 August 1996     

Characterization of Listeria monocytogenes isolates from food animal clinical cases: PFGE pattern similarity to strains from human listeriosis cases

Twenty-one isolates of Listeria monocytogenes from food animal clinical cases that involved meningitis or meningoencephalitis, encephalitis, mastitis and abortion were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) in order to improve our understanding of the genetic links between individual strains and strains recovered from human listeriosis cases. Results showed that five of the isolates were serotype 1/2a, six were 1/2b, nine were 4b, and one was untypeable. A caprine, two bovine and an ovine brain isolate shared identical PFGE patterns indicating that strains of L. monocytogenes are not host specific. Other isolates exhibited distinct patterns that were not shared, indicating a genetic diversity. Dendrogram analysis revealed that PFGE patterns of the isolates clustered primarily according to serotype. We compared the PFGE types obtained for these isolates with PFGE types for human clinical isolates present in the CDC national PulseNet database. Six (29%) of the twenty-one strains had patterns that were indistinguishable from pathogenic human isolates in the database. Our observations offer preliminary evidence that food animals could be significant reservoirs of L. monocytogenes that lead to human infections and support the inclusion of PFGE patterns of veterinary clinical isolates in the national PulseNet database for increased surveillance.     

Molecular typing of Salmonella weltevreden and Salmonella chincol by pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)

Genomic DNA of Salmonella weltevreden (10 isolates from poultry, two isolates each from raw vegetables and river water) and S. chincol (15 isolates from poultry) were characterized by pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) analysis. These isolates originated from a single location in Kajang, Selangor. The results of the PFGE and ERIC-PCR were analysed and comparisons were made using GelCompar software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the S. weltevreden into five clusters and two single isolates and S. chincol into two clusters and two single isolates at a similarity level of 80%, respectively. PFGE produced a single cluster and eight single isolates for S. weltevreden, and one cluster and 11 single isolates for S. chincol at a similarity level of 80% after digestion with the restriction enzyme XbaI, respectively. These results demonstrate that both PFGE and ERIC-PCR are suitable tools for molecular typing of the isolates examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Variation in sic Gene Encoding Complement-Inhibiting Protein of Streptococcus pyogenes Serotype M1 Isolates in Japan

DNA sequencing of the gene encoding a complement-inhibiting protein of Streptococcus pyogenes (streptococcal inhibitor of complement, Sic) was carried out on 49 strains of S. pyogenes serotype M1. Those strains were obtained from patients and asymptomatic carriers in Japan from 1969 to 1997 and had various pulsed-field gel electrophoresis (PFGE) patterns. Four identical polymorphic sites were found in the strains with the same PFGE pattern (Ia), but not in those giving the pattern IIa. The other identical sites were found in the strains with the PFGE pattern IIa, but not in those with the pattern Ia. These observations suggest that each of PFGE patterns was restricted to a set of variation in the sic gene. Received: 13 January 2000 / Accepted: 24 February 2000     

Molecular Characterization of Yarrowia lipolytica and Candida zeylanoides Isolated from Poultry

Yeast isolates from raw and processed poultry products were characterized using PCR amplification of the internally transcribed spacer (ITS) 5.8S ribosomal DNA region (ITS-PCR), restriction analysis of amplified products, randomly amplified polymorphic DNA (RAPD) analysis, and pulsed-field gel electrophoresis (PFGE). ITS-PCR resulted in single fragments of 350 and 650 bp, respectively, from eight strains of Yarrowia lipolytica and seven strains of Candida zeylanoides. Digestion of amplicons with HinfI and HaeIII produced two fragments of 200 and 150 bp from Y. lipolytica and three fragments of 350, 150, and 100 bp from C. zeylanoides, respectively. Although these fragments showed species-specific patterns and confirmed species identification, characterization did not enable intraspecies typing. Contour-clamped heterogeneous electric field PFGE separated chromosomal DNA of Y. lipolytica into three to five bands, most larger than 2 Mbp, whereas six to eight bands in the range of 750 to 2,200 bp were obtained from C. zeylanoides. Karyotypes of both yeasts showed different polymorphic patterns among strains. RAPD analysis, using enterobacterial repetitive intergenic sequences as primers, discriminated between strains within the same species. Cluster analysis of patterns formed groups that correlated with the source of isolation. For ITS-PCR, extraction of DNA by boiling yeast cells was successfully used.     

Changes in Pulsed-Field Gel Electrophoresis Patterns in Clinical Isolates of Enterohemorrhagic Escherichia coli O157:H7 Associated with Loss of Shiga Toxin Genes

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains showed disappearance of a 70- or 80-kb fragment in their patterns associated with loss of Shiga toxin genes during maintenance or subcultivation. Hybridization experiments with a DNA probe complementary to Shiga toxin sequences revealed that the Shiga toxin genes in the parental strain were located on fragments the same size as the lost fragments from the toxin-negative derivatives. The evidence indicates that PFGE pattern of EHEC O157:H7 may change due to loss of Shiga toxin genes, which is likely to be associated with curing of Shiga toxin gene carrying phages in vitro. Received: 4 May 1998 / Accepted: 19 August 1998     

DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.     

Evidence of Genomic Instability in Campylobacter jejuni Isolated from Poultry

Poultry isolates of Campylobacter jejuni derived from a survey of meat processing batches were genotyped by pulsed-field gel electrophoresis (PFGE) of chromosomal DNA to establish the clonal relationships between single-colony isolates. In the majority of batches studied, one or two genotype patterns predominated. However, in one batch (batch A), 21 single-colony isolates gave 14 different PFGE genotypes. The banding patterns obtained with SmaI were sufficiently different to distinguish between genotypes, although the patterns also produced many common bands. The question of whether these isolates represented different clones or had a common clonal ancestry was addressed by additional genotypic and phenotypic methods. Restriction length polymorphism of PCR products obtained from the flagellin genes showed an identical flagellin genotype for all of these isolates. In contrast, unrelated control isolates resulted in different flagellin genotypes. Moreover, all 14 different PFGE genotypes of batch A had identical Penner serotypes and identical or similar biotypes and phage types. It was concluded that the isolates were of clonal origin and that the diversity in the PFGE banding patterns had most likely originated from genomic rearrangements. However, the PFGE genotypes were shown to be stable upon subculturing in vitro and after in vivo passage in chickens, and natural transformation between isogenic mutants carrying antibiotic markers did not occur in vivo in a chick colonization model. The possible mechanisms for the hypothesized genomic recombinations and the conditions that allow, induce, or select for such events are discussed.     

Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

Southern blotting analyses of strains ofCampylobacter fetus using the conserved region ofsapA

Chromosomal DNA of 27 strains of Campylobacter fetus was analyzed by Southern blotting with a probe of the conserved region of sapA. The probe hybridized with 23 strains that produced type A lipopolysaccharide. These strains had more than six sapA homologs. In Southern blots of SalI-digested chromosomal DNA separated by pulsed-field gel electrophoresis, one fragment from 19 strains and two fragments from 4 strains hybridized. These data indicate that multiple sapA homologs are localized to a limited region on the chromosomal DNA of C. fetus and thus suggest the possibility of developing a typing system using this method. Received: 28 June 1995 / Accepted: 19 September 1995     

Analysis of pristinamycin-resistant Staphylococcus epidermidis isolates in the Tunisian Bone Marrow Transplant Center

Aims: We report the analysis of genetic determinants conferring resistance to pristinamycin in Staphylococcus epidermidis strains and epidemiology typing of these strains by pulsed‐field gel electrophoresis. Methods and Results: Staphylococcus epidermidis (346 isolates) were searched for strains with pristinamycin resistance. Pristinamycin‐resistant strains (seven isolates) were isolated in five patients with haematological cancer in the Bone Marrow Transplant Centre of Tunisia in 2002. Resistance to pristinamycin was observed in 2% of isolates. The seven pristinamycin‐resistant strains shared resistance to oxacillin (MIC = 8–512 μg ml^?1), gentamicin (MIC = 16–512 μg ml^?1), erythromycin (MIC > 1024 μg ml^?1), lincomycin (MIC > 1024 μg ml^?1), pristinamycin (MIC = 4–16 μg ml^?1) and rifampin (MIC = 128–256 μg ml^?1). erm genes were amplified: ermA from six strains and ermC from one. vga gene encoding streptogramins A resistance (pristinamycin résistance) was amplified from all strains and typed as vgaA by analysis after electrophoresis of restriction profiles of vga amplicons (two fragments with Sau3A of 164 and 378 bp; one fragment with EcoRI). Pulsed‐field gel electrophoresis (PFGE) of SmaI chromosomal DNA digests of the seven S. epidermidis isolates divided them into two distinct pattern types: pulsed‐field type A (classified from A1 to A6 subtypes) and type B. The six strains harbouring ermA genes belonged to the PFGE type A while the strain harbouring ermC genes belonged to the PFGE type B. We characterized an epidemic strain carrying the vgaA and ermA genes responsible for the outbreak. Conclusions: Two clones of pristinamycin‐resistant S. epidermidis were isolated in our patients. One of them, isolated in all patients, had expanded over six months suggesting acquisition by cross‐contamination. Significance and Impact of the study: Increasing isolation of pristinamycin resistant S. epidermidis strains is an alarming indicator of nosocomial dissemination. The vector will be determined to establish a system of epidemiological surveillance.     

Use of pulsed field gel electrophoresis as an epidemiological tool for analysis of sporadic associated strains of Salmonella typhi isolated in Taiwan

In order to characterize the subtypes of Salmonella typhi which cause sporadic disease in Taiwan, 55 isolates of Salm. typhi obtained from unrelated patients of sporadic cases during 1992-96 were subjected to chromosomal DNA digestion and pulsed field gel electrophoresis (PFGE). When DNAs of these 55 Salm. typhi strains were digested with XbaI, 41 PFGE patterns were observed. Strains sharing the same XbaI digestion pattern could not be further discriminated by PFGE analysis using SpeI and NotI as digestion enzymes. Thus, considerable genetic diversity exists among the Salm. typhi isolates. Although strains of the same patterns were mainly isolated during the same time, recirculation of certain infectious strains could be possible. When 12 antibiotics, i.e. ampicillin, trimethoprim/sulfamethoxazole, erythromycin, norfloxacin, tetracycline, sulphonamide, streptomycin, neomycin, chloramphenicol, kanamycin, cefoperazone and gentamycin were used to test the antibiotic susceptibility for these Salmonella isolates, only three antibiogram patterns were obtained and 49 of the 55 Salm. typhi isolates were found to belong to one pattern. Phage typing and plasmid profiles were also poor in discriminating these strains. Thus, PFGE alone may be used as a powerful tool for analysis of sporadic associated Salm. typhi strains.     

Physical and genetic chromosomal maps of Streptococcus agalactiae, serotypes II and III; rRNA operon organization

A detailed analysis of two Streptococcus agalactiae (group B streptococcus, GBS) strains was performed by pulsed field gel electrophoresis (PFGE). Digestion of the chromosomal DNA with SmaI and SgrAI endonucleases, followed by separation and analysis of fragments by PFGE was carried out. Physical chromosomal maps of serotype II/(α+β) and III/α strains of S. agalactiae were constructed. The GBS genome size was estimated to be 2200 kb. Sixteen GBS genes were used as probes and were located on the restriction maps of both strains by DNA-DNA hybridization. Six copies of ribosomal operons were found in the genome of the analyzed strains. Significant differences in the restriction patterns of chromosomal DNA and DNA-DNA hybridization between the two analyzed strains were detected so that DNA restriction patterns may be used to trace outbreaks of disease. The overall GBS chromosomal organization as determined is fairly conserved.     

Isolation and characterization of Salmonella enterica from Antarctic wildlife

In recent years, the human presence in Antarctica has increased and as a consequence, the possibility of microorganisms’ introduction. The aims of this work were to determine the presence of Salmonella enterica in Antarctic seabirds and sea mammals, to characterize the isolates identified, and to determine the genetic relation of Antarctic S. enterica isolates among them and compare with isolates of human, animal, and food sources recovered in Argentina. During the summer 2000 and 2002 in Potter Peninsula, and during the summer 2001 and 2003 in Hope Bay, a total of 1,739 fecal samples from Antarctic animals were collected and analyzed. In summer 2000, S. Newport and S. Enteritidis were isolated from 8.9% of southern giant petrels (Macronectes giganteus). In summer 2003, S. Enteritidis was isolated from 1.5% of Adelie penguins (Pygoscelis adeliae), from 5.5% of skuas (Stercorarius sp.), from 5.4% of kelp gulls (Larus dominicanus), and from 5.6% of Weddell seals (Leptonychotes weddelli). All the isolates belonging to the same serovar showed indistinguishable genomic profiles by Pulse-Field Gel Electrophoresis (PFGE) with XbaI and BlnI restriction enzymes and by Random Amplified Polymorphic DNA (RAPD-PCR). In addition, these Antarctic strains were different from S. enterica isolates from different sources identified in Argentina during the same or close time periods.     

Molecular differentiation of Erwinia amylovora strains from North America and of two Asian pear pathogens by analyses of PFGE patterns and hrpN genes

In order to determine a possible genomic divergence of Erwinia amylovora'fruit tree' and raspberry strains from North America, several isolates were differentiated by pulsed-field gel electrophoresis (PFGE) analysis, the size of short DNA sequence repeats (SSRs) and the nucleotide and deduced amino acid sequences of their hrpN genes. By PFGE analysis European strains are highly related, whereas strains from North America were diverse and were further distinguished by the SSR numbers from plasmid pEA29. The E. amylovora strains from Europe showed identical HrpN sequences in contrast to the American isolates from fruit trees and raspberry. Those were related to each other, but distinguishable by their HrpN patterns. The Asian pear pathogens differed in HrpN among each other and from E. amylovora. Erwinia pyrifoliae isolates and the Erwinia strains from Japan were ordered via their HrpN sequences in agreement with the PFGE patterns. For all three pathogens, dendrograms from PFGE and sequence data indicate an evolutionary diversity within the species in spite of a genetic conservation for parts of the hrpN genes suggesting a long persistence of the Asian pear pathogens in Korea and Japan as well as of fire blight in North America. Some of the divergent American E. amylovora isolates share PFGE patterns with the relatively uniform European strains.     

Comparison of Three Methods for Epidemiological Typing of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">C. coli</Emphasis>

A total of 168 Campylobacter strains (154 C. jejuni and 14 C. coli) isolated from human clinical samples and chicken meat were typed using Penner serotyping, randomly amplified polymorphic DNA (RAPD), and pulsed-field gel electrophoresis (PFGE) with four restriction enzymes (Sac II, Sal I, Sma I, Kpn I).The 168 strains were found to represent 13 different Penner-types and 72 different RAPD-types. However, the discriminatory potential of PFGE was dependent on the restriction enzymes used. The 168 strains were divided into 74 (Sac II), 73 (Sal I), 72 (Sma I) and 69 (Kpn I) types. The DNA of some strains was not digested by Sal I, Sma I and Kpn I. Although three RAPD-types were further subdivided by PFGE, RAPD showed good discriminatory power and a high level of agreement with PFGE patterns in terms of strain differentiation.To compare the similarities of PFGE patterns (Sac II) among the strains, a dendrogram was constructed based on the unweighted pair group method with averages (UPGMA). In most cases, DNA types of C. coli were different from those of C. jejuni. The similarities between human and meat isolates were less than 0.42 except for one outbreak in which the isolates from both patients and chicken meat showed the same DNA types.