Typing of toxic strains of Clostridium difficile using DNA fingerprints generated with arbitrary polymerase chain reaction primers

Clostridium difficile is the causative agent for pseudomembranous colitis in humans. Toxic strains of C. difficile produce two toxins, toxin A and toxin B. A reliable and definitive method of typing the toxic strains of C. difficile is needed since nosocomial cross infection is a primary concern in hospitals and other health care facilities. A method for typing toxic strains of Clostridium difficile using arbitrary polymerase chain reaction (PCR) primers is presented in this study. The C. difficile strains were initially characterized for the toxin A genetic determinant using specific PCR primers which differentiate toxin positive from toxin negative strains. These toxic strains were then PCR typed using six arbitrary primers which generated DNA patterns that were unique for all toxic strains examined. The use of this typing scheme in clinical applications is discussed.     

SYBR green real-time PCR method to detect Clostridium botulinum type A

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).     

Polymerase Chain Reaction Test for Differentiation of Five Toxin Types of Clostridium perfringens

In order to avoid the use of experimental animals, the polymerase chain reaction (PCR) method was applied to differentiate Clostridium perfringens into five toxin types. Twenty-two out of 23 strains tested produced the toxin(s) corresponding to the toxin gene(s) identified by PCR, and vice versa. Consequently, the gene typing was consistent with conventional typing by animal tests. Twenty-five strains were identified as types different from original ones by the PCR method as well as a toxin neutralization test. These findings suggest that the PCR method, which is easy and timesaving, is applicable to identify the toxin types of C. perfringens as an alternative to animal tests, and that beta-, epsilon- and iota-toxin genes might be lost by long-term preservation. The reasons why the strains lost the genes are discussed.     

Further development of a bacteriocin typing system for Clostridium perfringens

The specificity of typing Clostridium perfringens with bacteriocins was improved by adding new bacteriocins and deleting others from the original typing set of ten. A total of 516 new isolates of Cl. perfringens were screened for bacteriocin production and, of these, 162 strains (31%) were found to be producers. The sensitivity patterns obtained by testing 40 bacteriocins against 200 isolates of Cl. perfringens were recorded and the data subjected to a computer analysis. A total of 18 bacteriocins capable of dividing the 200 isolates into 98 typing patterns was selected. The repro-ducibility of the new system was tested by performing three sequential typings of 60 strains of Cl. perfringens. No variation was found in 73% of the strains, while a further 16% of the strains demonstrated a change in sensitivity to only one bacteriocin. Common serological types of Cl. perfringens were divisible into subtypes based upon both their ability to produce bacteriocins and their sensitivity to bacteriocins, suggesting a useful role for bacteriocin typing in conjunction with an already well-established tool for typing Cl. perfringens.     

High Molecular Weight Typing with MALDI-TOF MS - A Novel Method for Rapid Typing of Clostridium difficile

Clostridium difficile strains were typed by a newly developed MALDI-TOF method, high molecular weight typing, and compared to PCR ribotyping. Among 500 isolates representing 59 PCR ribotypes a total of 35 high molecular weight types could be resolved. Although less discriminatory than PCR ribotyping, the method is extremely fast and simple, and supports for cost-effective screening of isolates during outbreak situations.     

Typing of Clostridium perfringens strains by use of Random Amplified Polymorphic DNA (RAPD) system in comparison with zymotyping

The definition of strain clonality postulates that strains showed identical phenotypic and genetic traits are likely to descend from a common ancestor even if they were isolated from different sources and locations. Regarding this definition, non-epidemiologically linked strains might be clonal strains. To overcome this ambiguity, the discriminatory capability of RAPD typing was assessed firstly on eight Clostridium perfringens strains proven to be chromosomally different with one being the mutant of another one. Thirteen primers were tested but only two were able to differentiate seven of the eight strains. With none of the used primers it was possible to differentiate the parental strain and its mutant harboring an insertion of 180 kb. The four most discriminant primers were retained to determine the RAPD fingerprints of a further 20 previously zymotyped strains from which seventeen were unrelated. To compare the two typing systems, the zymotype of the eight chromosomally different strains was determined. Thus, the discriminatory index was calculated on the basis of 25 unrelated C. perfringens strains. This was 0.97 with RAPD typing and 0.99 with zymotyping. From these results we conclude that the RAPD typing which is less fastidious than zymotyping can be used as an epidemiological marker for C. perfringens.     

Typing of sheep clinical isolates and identification of enterotoxigenic Clostridium perfringens strains by classical methods and by polymerase chain reaction (PCR).

A simple procedure was developed to identify toxitypes of Clostridium perfringens of different origins. Ninety strains of C. perfringens were identified by classical bacteriological methods, typing of the strains was done by a seroneutralisation test on mice. Production of enterotoxin was tested and all strains were analysed by PCR using gene of toxin alpha, gene of toxin E, gene of toxin beta and gene of enterotoxin. Simple amplification (amplifying one gene), and duplex and triplex amplification (amplifying two and three genes simultaneously) were performed. In the conditions of the experiment, the PCR method has proved efficacious. The specificity and sensitivity are excellent and superior to those of the classical methods. The prophylaxis of enterotoxaemia in animals is achieved by vaccination, the PCR technique can thus become a first-choice tool for the identification and typing of the C. perfringens strains which initiate these diseases. In turn, this would simplify the development of vaccines adapted to the epidemiological situation.     

Bacteriocin Susceptibility of Clostridium perfringens: a Provisional Typing Schema

Ninety-four strains of Clostridium perfringens were examined for bacteriocin production. Bacteriocins produced by ten of these strains were selected for typing 274 cultures of C. perfringens. The bacteriocins were prepared by growing the producer strains in broth and precipitating the active principle from the supernatant fluids of centrifuged cultures with ammonium sulfate. All bacteriocins were titrated against a common indicator strain, adjusted to equivalent titers, and spotted onto blood agar plates seeded with the test organisms. Fifty different bacteriocin sensitivity patterns were observed. These patterns were organized into seven groups bearing some relationship, and the largest number of strains falling into any one pattern did not exceed 16% of the total strains tested. Ninety-nine percent of all isolates were typable. The new method should prove useful in studies where strains must be fingerprinted.     

Development and application of a multiple typing system for Clostridium difficile.

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Development and application of a multiple typing system for Clostridium difficile

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Relapses or reinfections: Analysis of a case of Clostridium difficile-associated colitis by two typing systems

Immunoblotting and pulsed-field gel electrophoresis of Clostridium difficile isolates were employed to differentiate reinfection by a newly acquired strain from relapse by an original strain in a 10-year-old patient with four episodes of C. difficile-associated colitis. Immunoblot typing demonstrated subserogroup K-1 of serogroup K for the first and second organisms, subserogroup A-1 of serogroup A for the third organism, and subserogroup G-4 of serogroup G for the fourth organism. PFGE analysis revealed consistent results with immunoblot analysis except that the strains from the fourth episode, whose DNA constantly degraded, were nontypable by this method. Five separate isolates of C. difficile from a specimen of each episode showed identical PFGE patterns, indicating that infections of multiple strains probably did not occur in this patient. These typing results suggested that the second episode after a 17-day course of vancomycin therapy represented a relapse by the strain causing the first episode, and that the third and fourth episodes after tapering vancomycin therapy were reinfections by other strains. Both immunoblot and PFGE typing systems are promising tools for analyzing recurrence of C. difficile infection. Received: 27 November 1995 / Revised: 1 January 1996 / Accepted: 22 March 1996     

Numerical analysis of electrophoretic protein patterns of Providencia alcalifaciens strains from human faeces and veterinary specimens

Twenty-five strains of Providencia alcalifaciens from various countries have been characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 15 from human faeces, one from duck faeces, one from a guinea-pig eye and eight from unknown sources. Also included, for reference purposes, were the type strains of three other Providencia species. The protein patterns, which contained 45–50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, in which the principal protein bands (in the 33–40 kD range) were excluded, the 25 Prov. alcalifaciens strains formed, at the 83% S level, a single cluster whilst the three Providencia reference strains remained unclustered. In the second, which included all the protein bands, the 25 Prov. alcalifaciens strains formed 10 clusters at the 85% S level. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov, alcalifaciens . Reference strains of each of the 10 PAGE types identified are available from NCTC for inclusion in future studies.     

Numerical analysis of electrophoretic protein patterns of Providencia alcalifaciens strains from human faeces and veterinary specimens

Twenty-five strains of Providencia alcalifaciens from various countries have been characterized by one-dimensional SDS-PAGE of cellular proteins. They comprised 15 from human faeces, one from duck faeces, one from a guinea-pig eye and eight from unknown sources. Also included, for reference purposes, were the type strains of three other Providencia species. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, in which the principal protein bands (in the 33-40 kD range) were excluded, the 25 Prov. alcalifaciens strains formed, at the 83% S level, a single cluster whilst the three Providencia reference strains remained unclustered. In the second, which included all the protein bands, the 25 Prov. alcalifaciens strains formed 10 clusters at the 85% S level. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. alcalifaciens. Reference strains of each of the 10 PAGE types identified are available from NCTC for inclusion in future studies.     

粪便标本艰难梭菌三种培养方法的比较

目的 比较粪便标本中艰难梭菌三种培养方法的差异性,寻找适合临床实验室使用的艰难梭菌培养方法。方法 将健康儿童的300份粪便标本分别用快速芽孢孵育法、乙醇直接处理法、富集芽孢培养法进行培养,并对三种培养方法的差异性进行统计学比较。结果 300份粪便标本快速芽孢孵育法培养出艰难梭菌51例,检出率为17.0%;乙醇直接处理法培养出48例,检出率为16.0%;富集芽孢培养法培养出44例,检出率为14.7%。三种方法的检出率差异无统计学意义（P=0.7352,P>0.05）。结论 乙醇直接处理法、快速芽孢孵育法简单快速,适合临床实验室进行艰难梭菌的快速培养;富集芽孢培养法杂菌少,适合大量艰难梭菌培养便于结果的观察及进一步的处理。     

Numerical analysis of electrophoretic protein patterns of Morganella morganii strains from faeces, wound, urine and other clinical sources

Sixty-five strains of Morganella morganii (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 13 were from stools (including one from a toucan), 13 from wounds, 11 from urine, five from blood (including one from a snake), five from the respiratory tract (four sputum, one lung), 12 from miscellaneous sources and six from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of 67 M. morganii cultures plus those of the type strains of seven Proteus and Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the M. morganii strains formed 21 clusters at the 91% S level. In the second analysis, in which the principal protein bands (in the 31.6—43.2 kDa range) were excluded, the 67 M. morganii cultures formed a single cluster at the 80% S level distinct from the seven Proteus and Providencia reference strains. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of M. morganii . Reference strains of each of the 21 PAGE types identified are available from NCTC for inclusion in future studies.     

Numerical analysis of electrophoretic protein patterns of Providencia rettgeri strains from human faeces, urine and other specimens

Thirty-one strains of Providencia rettgeri (mainly from humans) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries and comprised 14 from urine, eight from faeces, two from bile (plus one from the liver of a sheep), two from sputum, one from an insect pupa and three the sources of which were unknown. Also included, for reference purposes, were the type strains of the four other Providencia species. The protein patterns, which contained 45-50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 31 Prov. rettgeri strains formed 13 clusters at the 84% S level. In the second analysis, in which the principal protein bands (in the 33.3-41.3 kD range) were excluded, 29 of the 31 Prov. rettgeri strains formed a single cluster at the 81% S level, whilst the four Providencia reference strains remained unclustered. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. rettgeri. Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.     

Numerical analysis of electrophoretic protein patterns of Providencia rettgeri strains from human faeces, urine and other specimens

Thirty-one strains of Providencia rettgeri (mainly from humans) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries and comprised 14 from urine, eight from faeces, two from bile (plus one from the liver of a sheep), two from sputum, one from an insect pupa and three the sources of which were unknown. Also included, for reference purposes, were the type strains of the four other Providencia species. The protein patterns, which contained 45–50 discrete bands, were highly reproducible and were used as the basis for two numerical analyses. In the first, which included all the protein bands, the 31 Prov. rettgeri strains formed 13 clusters at the 84% S level. In the second analysis, in which the principal protein bands (in the 33.3–41.3 kD range) were excluded, 29 of the 31 Prov. rettgeri strains formed a single cluster at the 81% S level, whilst the four Providencia reference strains remained unclustered. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. rettgeri. Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.     

Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.     

Numerical analysis of electrophoretic protein patterns of Providencia stuartii strains from urine, wound and other clinical sources

Eighty-six strains of Providencia stuartii (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 52 were from urine, 11 from wounds, five from blood (one of these also from urine), four from ear infections, two each from faeces and sputum, one from 'alimentation'and nine from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of 46 Prov, stuartii strains (selected to represent the full range of protein pattern diversity) plus those of the type strains of the four other Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the Prov. stuartii strains formed 13 clusters at the 88% S level. In the second analysis, in which the principal protein bands (in the 33.8–40.7 kDa range) were excluded, 45 of the 46 Prov. stuartii strains formed a single cluster at the 82% S level, whilst the four Providencia reference strains remained unclustered. The 40 strains of Prov. stuartii not included in the cluster analysis were assigned to a protein type by calculating their similarity with the strains in the database used for the cluster analysis. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. stuartii . Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.     

Numerical analysis of electrophoretic protein patterns of Providencia stuartii strains from urine, wound and other clinical sources

Eighty-six strains of Providencia stuartii (mainly of human origin) were characterized by one-dimensional SDS-PAGE of cellular proteins. The strains came from various countries; 52 were from urine, 11 from wounds, five from blood (one of these also from urine), four from ear infections, two each from faeces and sputum, one from 'alimentation' and nine from unknown sources. The protein patterns, which contained 45 to 50 discrete bands, were highly reproducible. The patterns of 46 Prov. stuartii strains (selected to represent the full range of protein pattern diversity) plus those of the type strains of the four other Providencia species were used as the basis for two numerical analyses. In the first, which included all the protein bands, the Prov. stuartii strains formed 13 clusters at the 88% S level. In the second analysis, in which the principal protein bands (in the 33.8-40.7 kDa range) were excluded, 45 of the 46 Prov. stuartii strains formed a single cluster at the 82% S level, whilst the four Providencia reference strains remained unclustered. The 40 strains of Prov. stuartii not included in the cluster analysis were assigned to a protein type by calculating their similarity with the strains in the database used for the cluster analysis. We conclude that high resolution PAGE combined with computerized analysis of protein patterns provides the basis for typing clinical strains of Prov. stuartii. Reference strains of each of the 13 PAGE types identified are available from NCTC for inclusion in future studies.