Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains

Plasmid analysis of over 120 strains of Clostridium perfringens, isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6.2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.     

Characterization of atypical Aeromonas salmonicida isolates by ribotyping and plasmid profiling

A total of 38 strains of atypical Aeromonas salmonicida , three oxidase-negative but otherwise typical Aer. salmonicida , three typical Aer. salmonicida , and two reference strains, isolated from several countries and fish species were examined with respect to rRNA gene restriction patterns (ribotypes) and plasmid profiles. Most epidemiologically unrelated strains had different ribotypes, whereas isolates from the same outbreak were identical. All strains, except one, carried one or more large plasmids (> 55 kbp) and all strains, except two, additionally carried one or more smaller plasmids. Many strains isolated from the same outbreak showed different plasmid profiles although some plasmids were identical. The results suggest the existence of several atypical Aer. salmonicida. It also seems that ribotypes are stable properties for these bacteria while the plasmids are more labile.     

Plasmid profiles and randomly amplified polymorphic DNA analysis of Salmonella enterica serotype Enteritidis strains from outbreaks and sporadic cases in Turkey

The aim of the study was to investigate the characteristics of Salmonella serotype Enteritidis strains isolated from outbreaks and sporadic cases in Turkey by plasmid profiles and randomly amplified polymorphic DNA (RAPD) patterns. A total of 64 S. Enteritidis clinical strains were selected from the culture collection of the Enterobacteria Laboratory of Ankara University Medical School Department of Microbiology and Clinical Microbiology for molecular analysis using the plasmid profiles and RAPD method. Fifty-six isolates (88%) harbored one to four plasmids ranging in size from 2.5 to 100 kbp. 57 kbp plasmids were the most common plasmids, and forty-four strains (69%) carried 57 kbp plasmids alone or together with other plasmids. The outbreak strains carried the same plasmid profile: three plasmids sized 57, 40, 3.0 kbp. None of the strains analyzed displayed any RAPD bands with the primer OPB-17. By using primer p-1254, 42 strains (66%) were divided into fourteen RAPD patterns. Ten of the outbreak strains (77%) showed >80% similarity by cluster analysis program. Analysis of RAPD-PCR with primer p-1254 proved an easy, rapid and discriminative method complementing antibiogram and plasmid profiles in routine laboratories, and may contribute to the investigations of S. Enteritidis which still cause outbreaks in Turkey. This study presents the first report on S. Enteritidis isolates in Turkey investigated by plasmid profiles and RAPD methods.     

Distinct plasmid profiles of Pasteurella haemolytica serotypes and the characterization and amplification in Escherichia coli of ampicillin-resistance plasmids encoding ROB-1 beta-lactamase.

Thirty-five isolates of Pasteurella haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype A1, three of serotype A2 and one untypable) contained plasmid DNA and isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic animals or from animals in contact with pneumonic cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing A1 strains exhibited ampicillin resistance (ApR), which was shown by transfer studies to be plasmid-mediated. Plasmid DNA prepared from E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol (Cm) or by modifying the growth conditions. The ApR plasmids from P. haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 beta-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae. From substrate profiles and isoelectric focusing data, the beta-lactamases encoded by the P. haemolytica plasmids were indistinguishable from the ROB-1 beta-lactamase.     

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.     

Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes.

A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes. The results confirmed that V. anguillarum O1 is a biochemically homogeneous group. The 103 strains could be allocated to three main clusters with high similarity coefficients. None of the biochemical properties were connected with the presence of plasmids. In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme. Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish. Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids. All strains isolated from the kidneys of diseased fish on this farm had plasmids. Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1. These results suggest that pathogenic V. anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations. Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo.     

Genetic diversity of Pierce's disease strains and other pathotypes of Xylella fastidiosa

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.     

杭州市淋病奈瑟菌质粒酶切图谱分析研究

目的 :了解杭州市淋病奈瑟菌质粒携带及质粒谱型分布情况。方法 :采用碱裂解法对门诊 2 0 0 0年 1月～ 2 0 0 1年 10月分离的 2 0 7株淋病奈瑟菌进行了质粒抽提及质粒谱分型研究 ,并对菌株的青霉素耐药现象和耐药性质粒的关系进行探讨。结果 :2 0 7株淋病奈瑟菌中 194株 (93 .7% )检见质粒带 ,其中含一条质粒带的 112株 (5 4.11% ) ,含两条质粒带的 12株 (5 .8% ) ,含三条带的 70株 (3 3 .82 % ) ,尚有 13株(6.2 8% )未检测到质粒。以 E.coli V5 17细菌质粒作分子量标准 ,测得这些分子量分别为 2 .6、4.5、和 2 4.5Md。质粒谱型以 2 .6 4.5 2 4.5 Md(3 3 .82 % )多见。结论 :杭州地区质粒酶切图谱的分析研究有助于淋病的治疗和防治 ,这将对该地区淋病奈瑟菌的分子流行病学调查和淋病监控提供依据     

Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

Plasmids in Listeria monocytogenes in relation to cadmium resistance.

One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40% and 29%, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. monocytogenes have been shown to be associated with cadmium resistance.     

Plasmids in Listeria monocytogenes in relation to cadmium resistance.

One hundred and seventy-three unrelated Listeria monocytogenes strains isolated from humans, animals, the environment, and food were analyzed for the presence of plasmids. Extrachromosomal DNA was found in 28% of the strains. Plasmid DNA was extracted more frequently from L. monocytogenes serogroup 1 strains (35%) than from serogroup 4 strains (15%). Among strains from food and the environment, 40% and 29%, respectively, harbored plasmids, whereas only 13% of the strains from humans and animals with listeriosis bore plasmids. We also investigated the susceptibility of 90 strains to seven antibiotics and four heavy-metal salts. No antibiotic resistance could be detected, but 95.3% of the plasmid-positive strains and only 12.7% of the plasmid-negative strains were resistant to cadmium. The plasmid-determined genetic basis of cadmium resistance was proven by conjugation between strains of L. monocytogenes and by cure of the plasmid. This is the first time that plasmids of L. monocytogenes have been shown to be associated with cadmium resistance.     

Genetic Diversity of Pierce's Disease Strains and Other Pathotypes of Xylella fastidiosa

Strains of Xylella fastidiosa isolated from grape, almond, maple, and oleander were characterized by enterobacterial repetitive intergenic consensus sequence-, repetitive extragenic palindromic element (REP)-, and random amplified polymorphic DNA (RAPD)-PCR; contour-clamped homogeneous electric field (CHEF) gel electrophoresis; plasmid content; and sequencing of the 16S-23S rRNA spacer region. Combining methods gave greater resolution of strain groupings than any single method. Strains isolated from grape with Pierce's disease (PD) from California, Florida, and Georgia showed greater than previously reported genetic variability, including plasmid contents, but formed a cluster based on analysis of RAPD-PCR products, NotI and SpeI genomic DNA fingerprints, and 16S-23S rRNA spacer region sequence. Two groupings of almond leaf scorch (ALS) strains were distinguished by RAPD-PCR and CHEF gel electrophoresis, but some ALS isolates were clustered within the PD group. RAPD-PCR, CHEF gel electrophoresis, and 16S-23S rRNA sequence analysis produced the same groupings of strains, with RAPD-PCR resolving the greatest genetic differences. Oleander strains, phony peach disease (PP), and oak leaf scorch (OLS) strains were distinct from other strains. DNA profiles constructed by REP-PCR analysis were the same or very similar among all grape strains and most almond strains but different among some almond strains and all other strains tested. Eight of 12 ALS strains and 4 of 14 PD strains of X. fastidiosa isolated in California contained plasmids. All oleander strains carried the same-sized plasmid; all OLS strains carried the same-sized plasmid. A plum leaf scald strain contained three plasmids, two of which were the same sizes as those found in PP strains. These findings support a division of X. fastidiosa at the subspecies or pathovar level.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Large plasmids in an actinomycete

Abstract Using a modified procedure large indigenous plasmids were detected in cells of three strains belonging to a group of phenotypically similar actinomycetes isolated from the rhizoplane and root nodules of Alnus spp. M _r values of the plasmids were estimated to be about 80 · 10⁶ and 120 · 10⁶. The plasmid profiles of different strains of the group were found to be almost identical. This remarkable plasmid similarly is discussed in relation to the common source of isolation.     

Virulence studies based on plasmid profiles of the fish pathogen Vibrio salmonicida.

Strains of Vibrio salmonicida isolated from Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) suffering from cold-water vibriosis could be divided on the basis of plasmid profiles into four different categories. Of 32 strains, 19% harbored three plasmids of 24, 3.4, and 26 megadaltons (MDa), 69% harbored the 24- and 3.4-MDa plasmids but not the 2.6-MDA plasmid, and 9% harbored only the 24-MDA plasmid. The fourth category, which consisted of only one strain, harbored a plasmid of 10 MDa. In spite of different plasmid patterns, the strains of V. salmonicida were very similar with respect to biochemical reactions. The one-third of the V. salmonicida strains which were serotyped were of the same type. The 50% lethal doses, which were determined by intraperitoneal injection, ranged from 4 x 106 to 1 x 108 CFU per fish.     

Plasmids of serogroup 1 strains ofLegionella pneumophila

Twelve strains ofLegionella pneumophila were tested for the presence of plasmid DNA. Three strains, belonging to serogroup 1, had large plasmids of 83.8×10⁶ daltons, as determined by electron microscopy. A fourth strain, also from serogroup 1, had a similar large plasmid in addition to a smaller plasmid. Restriction analysis of plasmid DNA isolated from the strains with a single size plasmid indicated that the plasmids were structurally very similar. The biologic functions of these plasmids are yet to be determined.     

Characterization of plasmids that confer inducible resistance to 14-membered macrolides and streptogramin type B antibiotics in Staphylococcus aureus

During a period from 1978 to 1989, 413 Staphylococcus aureus strains were isolated at 27 different geographical regions in Hungary; they exhibited an inducible resistance to the 14-membered macrolides and streptogramin type B antibiotics, but not to the 16-membered macrolides and lincosamides: this resistance is referred to as PMS resistance phenotype. The isolates were mostly associated with patients suffering from staphylococcal diseases and with hygienic screenings in hospitals and closed communities. They were rarely isolated from food-poisoning cases, food hygienic screenings, or animal sources. Strains with PMS resistance phenotype were resistant to penicillin (99.0%), tetracycline (78.7%), and chloramphenicol (63.0%); however, they were susceptible to oxacillin. Most of them (94.2%) belonged to the phage type 52-complex. The determinant for PMS phenotype was located on plasmids, which also encoded beta-lactamase production and cadmium ion resistance, but not arsenate resistance. Three types of plasmid with molecular size of 50 kilobases (kb), 23.8 kb, and 16.8 kb, were found among the strains with PMS resistance phenotype, and the 50 kb and 23.8 kb plasmids also encoded mercury resistance. The 16.8 kb and 23.8 kb plasmids belonged to incompatibility group 1.     

Plasmid profiles of Staphylococcus aureus causing bovine mastitis

A. B aumgartner , J. N icolet & M. E ggimann 1984. Plasmid profiles of Staphylococcus aureus causing bovine mastitis. Journal of Applied Bacteriology 56 , 159–163.
Plasmid profiles of Staphylococcus aureus , isolated from herds affected with chronic bovine mastitis, are heterogeneous, as shown by the study of 85 strains from 18 different farms. The strains isolated within a herd sometimes show related plasmid patterns, although even strains isolated from different quarters of the same udder may show variations in their plasmid content. Strains without antibiotic resistance are frequently free of plasmids. Therefore, the existence of mastitis Staph. aureus virulence plasmids is unlikely. Staphylococcus aureus , resistant to penicillin and streptomycin from 9 different farms show related plasmid patterns. This result confirms a geographic spread of certain mastitis Staph. aureus strains.     

Survey of the plasmid content of group A streptococci

Abstract Examination of 70 M-prototype group A streptococci showed small plasmids (2.0–2.5 MDa) to be present in strains representative of M-types 28, 57, 61, 63, 64 and 69. Identical results were obtained from M _r and restriction endonuclease analyses of a 2.2-MDa plasmid (pDN691) found in the M-type 69 strain and similar plasmids in the M57 prototype and two other independently isolated M-type 57 strains. In all four strains the presence of plasmid correlated with the production of bacteriocin-like inhibitory activity identifiable as P type 614. Similar analysis revealed a possible relationship between a 2.5-MDa plasmid in the prototype M61 and M64 strains and the production of P-type 216 inhibitory activity. A survey of 56 group A streptococci recovered in association with either rheumatic fever or nephritis failed to demonstrate plasmid DNA with the exception of 2.2-MDa plasmids in four nephritis-associated M-type 57 isolates.     

Evidence for genetic exchange and recombination of Rhizobium symbiotic plasmids in a soil population

A soil population of 16 Rhizobium leguminosarum bv. trifolii isolates was characterized by using three Sym (for symbiotic) plasmid-specific DNA hybridization probes: (i) an R. leguminosarum bv. trifolii-specific, repeated-sequence probe; (ii) a nifHDK gene probe, and (iii) a nod gene probe. A predominant Sym plasmid family was identified among the isolates. Three other unrelated Sym plasmid families were also identified. The isolates were also classified either by using a chromosomal DNA hybridization probe or by serological relatedness to 25 different R. leguminosarum bv. trifolii antisera. With either method, it was possible to group the 16 soil isolates into identical or related families. However, the correlation between the two techniques was not high. Irrespective of the means used to classify the bacterial host strain, it was possible to identify the same Sym plasmids in unrelated strains, as well as unrelated Sym plasmids in identical host strains. These data indicate that, within this soil population, there has been genetic exchange of Sym plasmids, and in one instance the hybridization pattern indicates that in vivo recombination of two different Sym plasmids may have occurred. Symbiotic effectiveness tests on red, strawberry, and subterranean clovers clearly differentiated the isolates. In general, the pattern of response was similar within groupings on the basis of Sym plasmid and chromosomal profiles but different between such groups.