Restriction Endonuclease Analysis Typing Research Articles

403. Strain Epidemiology of Clostridioides difficile across Three Geographically Distinct Medical Centers in Chicago

Abstract Background Clostridioides difficile infections (CDI) are caused by a large and diverse group of strains with differences in prevalence and associated morbidity. Over the past 20 years the C. difficile (CD) molecular epidemiology has changed as the prevalence of the epidemic strain recognized as restriction endonuclease analysis (REA) group BI or PCR-Ribotype group (RT) 027 has decreased. The objective of this study was to determine the current epidemiology of CD in the city of Chicago. Methods Baseline characteristics and symptoms were compared for 81 patients who tested positive for CD by PCR (tcdB) between 9/1/2021 and 10/7/2021 at 3 hospitals in the city of Chicago. Patients were classified as having healthcare-associated CDI (HA-CDI) if symptoms began >72 hours after hospital admission, community-associated CDI (CA-CDI) if symptoms began ≤72 hours prior to admission, and community-onset healthcare-associated CDI (COHA-CDI) if they had been hospitalized ≤4 weeks prior to CDI diagnosis. Available stools were cultured and recovered CD isolates underwent REA typing. Determination of CD colonization was made by review of symptoms including chronicity of symptoms, stool frequency, and response to treatment. Results Among all patients, 33% (27/81) were CA-CDI, 28% (23/81) COHA-CDI, 11% (9/81) HA-CDI, and 27% (22/81) were classified as colonized. Primary CDI accounted for 66% (39/59) of the infections. Among patients with a primary CDI, 46% (18/39) of patients were classified as CA-CDI whereas COHA-CDI and HA-CDI accounted for 54% (21/39) of infections. REA group Y was the most common group strain accounting for 29% (22/75) of isolates. (Figure 1) REA group Y accounted for 26% (7/27) of CA-CDI compared to 0 REA group BI [p=0.06], and REA group Y accounted for 35% (7/20) of all colonized patients. (Figure 2) Figure 1Figure 2 Conclusion There has been a marked change in the CD epidemiology within the city of Chicago since 2009 when REA group BI accounted for 61% of CDI (Black et al ICHE 2011; 32:897-902). REA group Y (typically identified as RT 014/020) is now the most common group strain in Chicago supplanting REA group BI (RT027). REA group Y appears to be associated primarily with CA-CDI and CD colonization. A detailed genomic analysis of REA group Y is required to determine potential reservoirs of REA group Y. Disclosures Mary K. Hayden, MD, Sanofi: Member, clinical adjudication panel Nicholas M. Moore, PhD, D(ABMM), Abbott Molecular: Grant/Research Support|Cepheid: Grant/Research Support Amanda Harrington, PhD, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc.|bioMeriuex/BioFire: Grant/Research Support Dale N. Gerding, MD, Destiny Pharma plc.: Advisor/Consultant Stuart Johnson, M.D., Ferring Pharmaceuticals: Membership on Ferring Publication Steering Committee|Ferring Pharmaceuticals: Employee|Summit Plc: Advisor/Consultant.

Association between Clostridioides difficile ribotypes, restriction endonuclease analysis types, and toxin gene expression

ObjectivesClostridioides difficile strains cause severe infection. Previous studies suggested that the virulence of C. difficile is dependent on ribotype; however, this hypothesis is still controversial. We aim to investigate the relationship between ribotypes, restriction endonuclease analysis (REA) types, and toxin gene expression in C. difficile strains. MethodsWe utilized 53 clinical C. difficile strains. All strains were assigned a molecular strain type using PCR ribotyping and REA typing and classified into 17 ribotypes and six REA types. The expression of toxin genes (tcdA, tcdB, and cdtB) in C. difficile strains were quantified by real-time PCR using each specific primer set, and expression was normalized to that of the housekeeping gene rpoA. ResultsAll 53 strains expressed tcdB and four strains expressed cdtB. Five strains did not express tcdA. Most ribotype and REA type strains expressed tcdA and tcdB similar to the BAA-1870 strain. In cdtB-positive strains, the cdtB expression levels were similar to those in the BAA-1870 strain. tcdA and tcdB expression levels were similar in the cdtB-positive and cdtB-negative strains. ConclusionToxin gene expression was not associated with the ribotype. Production of binary toxin C. difficile transferase was not related to tcdA and tcdB expression levels.

Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains

Restriction endonuclease analysis (REA) and PCR ribotyping are two typing systems that have been frequently utilized for molecular epidemiologic characterization of Clostridioides (Clostridium) difficile. To correlate typing data obtained from each method, we performed both REA and PCR ribotyping on a large and diverse set of historical and contemporary C. difficile infection clinical isolates. Eighty isolates were selected from each reference laboratory in the United States (Microbiology Reference Laboratory, Hines VA Medical Center) and United Kingdom (Clostridium difficile Network for England and Northern Ireland laboratory, University of Leeds). The 160 isolates were assigned to 82 unique ribotypes and 51 unique REA groups (116 unique REA types). In general, concordance between typing methods was good. Dendrogram analysis of PCR ribotype band patterns demonstrated close genetic relationships among strain types with discordant REA and ribotype assignments. While REA typing was more discriminatory, several REA types in this study were further discriminated by PCR ribotyping, indicating that discriminatory value of these typing methods may be strain dependent. These data will assist with molecular epidemiologic surveillance of strains identified by these two commonly used C. difficile typing systems.

Restriction Endonuclease Analysis Typing of Clostridium difficile Isolates.

Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.

U.S.-Based National Sentinel Surveillance Study for the Epidemiology of Clostridium difficile-Associated Diarrheal Isolates and Their Susceptibility to Fidaxomicin.

In 2011 a surveillance study for the susceptibility to fidaxomicin and epidemiology of Clostridium difficile isolates in the United States was undertaken in seven geographically dispersed medical centers. This report encompasses baseline surveillance in 2011 and 2012 on 925 isolates. A convenience sample of C. difficile isolates or toxin positive stools from patients were referred to a central laboratory. Antimicrobial susceptibility was determined by agar dilution (CLSI M11-A8). Clinical and Laboratory Standards Institute (CLSI), Food and Drug Administration, or European Union of Clinical Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied where applicable. Toxin gene profiles were characterized by multiplex PCR on each isolate. A random sample of 322 strains, stratified by institution, underwent restriction endonuclease analysis (REA). The fidaxomicin MIC90 was 0.5 μg/ml for all isolates regardless of REA type or toxin gene profile, and all isolates were inhibited at ≤1.0 μg/ml. By REA typing, BI strains represented 25.5% of the isolates. The toxin gene profile of tcdA, tcdB, and cdtA/B positive with a tcdC 18-bp deletion correlated with BI REA group. Moxifloxacin and clindamycin resistance was increased among either BI or binary toxin-positive isolates. Metronidazole and vancomycin showed reduced susceptibility (EUCAST criteria) in these isolates. Geographic variations in susceptibility, REA group and binary toxin gene presence were observed. Fidaxomicin activity against C. difficile isolated in a national surveillance study did not change more than 1 year after licensure. This analysis provides baseline results for future comparisons.

Clostridium difficile Outbreak Strain BI Is Highly Endemic in Chicago Area Hospitals

Describe the clinical and molecular epidemiology of incident Clostridium difficile infection (CDI) cases in Chicago area acute healthcare facilities (HCFs). Laboratory, clinical, and epidemiologic information was collected for patients with incident CDI who were admitted to acute HCFs in February 2009. Stool cultures and restriction endonuclease analysis typing of the recovered C. difficile isolates was performed. Two hundred sixty-three patients from 25 acute HCFs. Acute HCF rates ranged from 2 to 7 patients with CDI per 10,000 patient-days. The crude mortality rate was 8%, with 20 deaths occurring in patients with CDI. Forty-two (16%) patients had complications from CDI, including 4 patients who required partial, subtotal, or total colectomy, 3 of whom died. C. difficile was isolated and typed from 129 of 178 available stool specimens. The BI strain was identified in 79 (61%) isolates. Of patients discharged to long-term care who had their isolate typed, 36 (67%) had BI-associated CDI. Severe disease was common and crude mortality was substantial among patients with CDI in Chicago area acute HCFs in February 2009. The outbreak-associated BI strain was the predominant endemic strain identified, accounting for nearly two-thirds of cases. Focal HCF outbreaks were not reported, despite the presence of the BI strain. Transfer of patients between acute and long-term HCFs may have contributed to the high incidence of BI cases in this investigation.

Molecular Epidemiology ofClostridium difficileover the Course of 10 Years in a Tertiary Care Hospital

The molecular epidemiology of endemic and outbreak Clostridium difficile strains across time is not well known. HindIII restriction endonuclease analysis (REA) typing was performed on available clinical C. difficile isolates from 1982 to 1991. The annual incidence of C. difficile infection (CDI) ranged from 3.2 to 9.9 cases per 1000 discharges and was significantly higher in 1982, 1983, 1985, and 1991 (high-incidence years) than in other years (mean standard deviation number of cases for the high- vs the low-incidence years, 121.8 +/-20.4 and 70.0 +/-15.0; P =.002). A total of 696 (76.6%) of 908 C. difficile isolates were available for REA typing over the 10-year period. Large clusters (>or=10 CDI cases in consecutive months) were caused by REA types B1 and B2 in 1982 and 1983, F2 and B1 in 1985, and K1 in 1991 (high-incidence years). Small clusters of 4-9 CDI cases in consecutive months were caused by REA types G1 (1984), Y4 and Y6 (1987), Y2 (1988), L1 (1989), Y1 (1990), and K1 (1991). Current epidemic REA group BI (unrelated to type B1) was isolated 6 times, twice in 1984, 1988, and 1990. Years with a high incidence of CDI were associated with large clusters of specific REA types that changed yearly. The molecular epidemiology of CDI in this hospital was characterized by a wide diversity of C. difficile types and an ever-changing dominance of specific C. difficile types over time. The current epidemic BI group was found sporadically on 6 occasions. A changing CDI molecular epidemiology should be expected in the future.

The A, B, BI, and Cs ofClostridium difficile

The study by Belmares et al (1) is the most comprehensive, longitudinal study of the molecular epidemiology of Clostridium difficile infection to date. This study is impressive not only because restriction endonuclease analysis typing was performed on three-quarters of C. difficile isolates from 908 cases of C. difficile infection (CDI) at the Minneapolis Veterans Administration Medical Center (MVAMC) collected over a 10 year period, but also because the investigators recognized the importance of conducting this study despite the 18 year time span from the collection of the C. difficile isolates to publication. This study confirms what other less systematic and/or comprehensive studies have suggested, the molecular epidemiology of C. difficile in healthcare facilities is frequently both diverse and dynamic (2–8). This study must be interpreted in conjunction with knowledge of the other studies performed and activities that took place during the same time period at the MVAMC that may have affected C. difficile transmission (9–15). Infection control policies, changes in healthcare delivery, and interventions that took place over the 10 year period included: 1) placing patients with CDI in private rooms only if incontinent and unable to use a bedpan, 2) using a quaternary ammonium disinfectant for cleaning all hospital rooms, 3) authorizing nurses to send diarrheal stool for C. difficile testing without a physician order, 4) restricting the use of clindamycin starting September 1985, 5) donning gloves as the standard for handling all body fluids starting April 1988, and 6) moving all patients into a new building in June 1988. Although this study contributes significantly to our knowledge of C. difficile, it also highlights the need for more studies combining molecular epidemiology with CDI prevention efforts in order to understand the effectiveness of CDI prevention practices.

Distribution of Clostridium difficile strains from a North American, European and Australian trial of treatment for C. difficile infections: 2005–2007

Clostridium difficile is a widely distributed pathogen with multiple strain types as determined by restriction endonuclease analysis (REA) and by PCR ribotyping, two well-characterized typing systems. In this study, REA typing was performed on 894 C. difficile isolates from patients enrolled from 16 countries on three continents in two large, recently conducted clinical treatment trials of C. difficile infection. REA group BI (Ribotype 027) isolates were the most common strains identified and were widely distributed throughout North America, but restricted to three of thirteen countries in Europe. REA group J (Ribotype 001) isolates were the most common strains identified in Europe and non-specific REA groups (historically less frequent) were the most common strains identified in Australia. REA groups BI, J, G and CF correlated with specific PCR ribotypes whereas more than one ribotype was found within REA groups Y, BK, and K. International surveillance of C. difficile strains is important to document the changing epidemiology of this enteric pathogen that continues to cause healthcare facility outbreaks and sporadic infections in other settings.

Rifampin and Rifaximin Resistance in Clinical Isolates of Clostridium difficile

Rifaximin, a poorly absorbed rifamycin derivative, is a promising alternative for the treatment of Clostridium difficile infections. Resistance to this agent has been reported, but no commercial test for rifaximin resistance exists and the molecular basis of this resistance has not been previously studied in C. difficile. To evaluate whether the rifampin Etest would be a suitable substitute for rifaximin susceptibility testing in the clinical setting, we analyzed the in vitro rifaximin susceptibilities of 80 clinical isolates from our collection by agar dilution and compared these results to rifampin susceptibility results obtained by agar dilution and Etest. We found rifaximin susceptibility data to agree with rifampin susceptibility; the MICs of both antimicrobials for all isolates were either very low or very high. Fourteen rifaximin-resistant (MIC, > or = 32 microg/ml) unique isolates from patients at diverse locations in three countries were identified. Molecular typing analysis showed that nine (64%) of these isolates belonged to the epidemic BI/NAP1/027 group that is responsible for multiple outbreaks and increased disease severity in the United Kingdom, Europe, and North America. The molecular basis of rifaximin and rifampin resistance in these isolates was investigated by sequence analysis of rpoB, which encodes the beta subunit of RNA polymerase, the target of rifamycins. Resistance-associated rpoB sequence differences that resulted in specific amino acid substitutions in an otherwise conserved region of RpoB were found in all resistant isolates. Seven different RpoB amino acid substitutions were identified in the resistant isolates, which were divided into five distinct groups by restriction endonuclease analysis typing. These results suggest that the amino acid substitutions associated with rifamycin resistance were independently derived rather than disseminated from specific rifamycin-resistant clones. We propose that rifaximin resistance in C. difficile results from mutations in RpoB and that rifampin resistance predicts rifaximin resistance for this organism.

Investigations on the clonality of isolates of Pasteurella gallinarum obtained from turkeys in Germany

Pasteurella gallinarum has been considered an opportunistic pathogen rather than a primary pathogen for chickens. As P. gallinarum has been found to have a high genotypic diversity, one would expect a polyclonal distribution among isolates from different farms if this organism is a secondary invader. The aims of this investigation were to genetically characterize isolates obtained from outbreaks affecting several turkey farms to confirm the existence of the infection in turkeys and to investigate the genetic relationship between isolates from affected farms. A total of 17 isolates from 14 outbreaks of respiratory disease in Germany were subjected to extended phenotypic and genotypic characterization. All isolates were of the same phenotype, typical of P. gallinarum. Ribotyping of three isolates using either HpaII or HindIII showed that they had identical profiles and indicated that the isolates all originated from the same clone. Comparison with HpaII ribotypes from a previous study showed that the pattern was identical to that obtained with isolates from a Zimbabwean outbreak in chickens during 1999 to 2000. Restriction endonuclease analysis typing of 14 isolates from all 14 farms showed that they had identical profiles but these differed from those obtained with isolates from the Zimbabwean outbreak. Sequencing of the 16S rRNA gene and sequence comparisons with other Pasteurellaceae confirmed their classification as P. gallinarum. Identification of the same clone of P. gallinarum from 14 outbreaks of acute respiratory disease in turkeys within a time period of 2 months suggests a common source of infection, and that P. gallinarum probably played a primary role rather than a secondary role in the outbreaks.

Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins.

Pathogenic strains of Clostridium difficile commonly produce two large clostridial toxins (LCTs), A and B, virulence factors responsible for C. difficile disease. Some strains have been reported to produce an additional toxin, a binary toxin designated CDT. Binary toxin has cytotoxic effects on cells in culture, but its role in human disease is not yet defined. In this study we examined the frequency of binary toxin genes (cdtB and cdtA) among C. difficile isolates that do not produce LCTs (A(-) B(-)) from a large United States-based collection organized by restriction endonuclease analysis (REA) typing. Of 58 strains tested, 9 (15.5%) were cdtB and cdtA positive, including 4 of 46 (8.7%) non-LCT-producing REA groups, with an estimated prevalence of at least 2% of all non-LCT-producing isolates within the collection. Five of the binary toxin-positive strains belonged to toxinotype XI, which does not produce LCTs but has minor parts of the LCT coding region or pathogenicity locus (PaLoc). We describe two new binary toxin-positive variants, one without any remnant of the LCT genes. This previously unknown variation was found in three isolates that were unrelated by REA typing. LCT-negative, binary toxin-positive strains were isolated from symptomatic and asymptomatic patients and from the hospital environment.

Molecular epidemiology of vancomycin-resistant enterococci: a 2-year perspective.

To determine the molecular epidemiology of vancomycin-resistant enterococci (VRE) at our medical center in order to identify the extent of strain clonality and possible transmission patterns of this pathogen. An important facet of our infection control program includes molecular typing of all clinical and surveillance isolates of VRE to determine transmission patterns in the hospital. Molecular strain typing is performed by restriction endonuclease analysis (REA) of genomic DNA. REA patterns are visually compared to categorize VRE strains into type and subtype designations. A 588-bed, university-affiliated, tertiary-care hospital and a neighboring 155-bed rehabilitation facility. From January 1995 through December 1996, 379 VRE isolates were collected from 197 patients. Thirty-three genotypes were determined by REA typing; 15 genotypes were implicated in 29 instances of potential nosocomial transmission. Three major clusters of VRE involving patients on multiple nursing units and two adjacent hospitals were identified. The remaining instances of nosocomial transmission occurred in small patient clusters. In conclusion, the VRE epidemic at this medical center is polyclonal. VRE transmission patterns are complex, and, while large clusters do occur, the usual pattern of nosocomial acquisition of this pathogen occurs in the setting of "mini-clusters".

Acquisition of Clostridium difficile and Clostridium difficile-associated diarrhea in hospitalized patients receiving tube feeding.

Clostridium difficile is the most common infectious cause of nosocomial diarrhea, but its role in diarrhea associated with tube feeding has not been rigorously investigated. To determine the incidence of C. difficile acquisition and C. difficile-associated diarrhea in tube-fed and non-tube-fed patients. Prospective cohort study. A university-affiliated Veterans Affairs Medical Center. 76 consecutive hospitalized, tube-fed patients and 76 hospitalized, non-tube-fed patients. The two cohorts were matched for age, unit location, duration of hospitalization before surveillance, and severity of illness. Incidence of C. difficile acquisition, incidence of C. difficile-associated diarrhea, and C. difficile restriction endonuclease analysis typing results. More tube-fed patients than non-tube-fed patients acquired C. difficile (15 of 76 patients [20%] compared with 6 of 76 patients [8%]; P=0.03) and developed C. difficile-associated diarrhea (7 of 76 patients [9%] compared with 1 of 76 patients [1%]; P=0.03). The mean proportion (+/-SD) of surveillance days with diarrhea was greater for tube-fed patients after the development of C. difficile-associated diarrhea than for tube-fed patients without this diarrhea (0.68+/-0.4 compared with 0.22+/-0.2 [95% CI for the mean difference, 0.08 to 0.84]). Postpyloric tube feeding (odds ratio, 3.14 [CI, 1.008 to 9.77]) and duration of surveillance (odds ratio, 1.08 [CI, 1.0009 to 1.16]) were risk factors for the acquisition of C. difficile. Nineteen restriction endonuclease analysis types of C. difficile were identified from 20 patients. Hospitalized, tube-fed patients, especially those receiving postpyloric tube feeding, are at greater risk for the acquisition of C. difficile and the development of C. difficile-associated diarrhea than are hospitalized, non-tube-fed patients. Clinicians should test for C. difficile in tube-fed patients with diarrhea.

Computerized restriction endonuclease analysis compared with O-serotype and phage type in the epidemiologic fingerprinting of Pseudomonas aeruginosa strains.

OBJECTIVE: To assess restriction endonuclease analysis (REA) of chromosomal DNA using SalI enzyme, low-concentration (0.4%) agarose gels and digitalized data management of the REA patterns obtained for the typing of clinical Pseudomonas aeruginosa isolates. METHODS: A group of 67 clinical unrelated isolates from 10 Spanish hospitals was used to study the discriminatory power, reproducibility and typeability of REA typing. RESULTS: A SalI REA pattern consisted of a variety (1--10) of restriction bands in the range between 12.2 and 48.5 kb and an unresolvable smear of low-molecular-weight bands. Forty different SalI REA patterns with an index of discrimination of 0.979 were obtained. Low typeability (91.04%) was the major limitation of REA typing. Analysis of blinded subcultures of eight Pseudomonas aeruginosa strains showed the reproducibility of REA typing to be 87.5%. Combined phenotypic typing (O-serotyping and phage typing) performed on the same group of strains showed comparable discrimination but much lower reproducibility. Isolates selected from five clusters of nosocomial infections in hospitals in the UK were typed by REA typing, and the results show high agreement when compared with conventional phenotypic typing methods in distinguishing between strains. CONCLUSIONS: These data underline the usefulness of REA typing enhanced with digitalized data management for the epidemiologic subtyping of clinical Pseudomonas aeruginosa isolates.

Investigation of Candida albicans transmission in a surgical intensive care unit cluster by using genomic DNA typing methods

An apparent outbreak of serious Candida albicans infections (n = 6) occurred in a surgical intensive care unit over a 4-week period. Four patients developed C. albicans bloodstream infections. An additional patient developed catheter-related C. albicans infection; the sixth patient developed an infection of cerebrospinal fluid. C. albicans was isolated from the hands of five health care workers (17%) and the throat of one health care worker (3%) during the outbreak investigation. Karyotyping and restriction endonuclease analysis of genomic DNA with BssHII of 23 C. albicans isolates from patients and the 6 health care worker isolates revealed 9 and 12 different patterns, respectively. Three of six patients appeared to be infected with the same C. albicans strain (two bloodstream infections and one cerebrospinal fluid infection). The hands of a health care worker were colonized with strain that appeared identical to an isolate from a patient prior to infection of the patient. However, restriction endonuclease analysis with SfiI found differences among the isolates determined to be identical by the other two methods. Karyotyping alone does not appear to be sufficient to differentiate between outbreak and control isolates. Restriction endonuclease analysis typing may be a more sensitive method than karyotyping alone in the investigation of a cluster of C. albicans infections. Furthermore, the use of more than one restriction enzyme may be necessary for optimal strain discrimination in restriction endonuclease analysis of genomic DNA.

Development of a rapid and efficient restriction endonuclease analysis typing system for Clostridium difficile and correlation with other typing systems

A HindIII restriction endonuclease analysis (REA) typing system for total genomic Clostridium difficile DNA including a rapid and efficient method of DNA extraction and a scheme for organizing unique electrophoretic DNA band patterns was developed. REA typing was performed by two extraction methods for 1,965 C. difficile isolates obtained from patients with symptomatic C. difficile disease, asymptomatic patients who were C. difficile culture positive, and environmental surfaces. This isolate collection yielded 206 unique REA types, which were organized into 75 groups. A reference strain representing each unique REA type was chosen for DNA band pattern comparisons, cytotoxin testing, and plasmid analysis. The DNA band patterns utilizing a guanidine thiocyanate-EDTA-Sarkosyl DNA extraction method were 94% reproducible, while the original and sporadically problematic diethyl pyrocarbonate-sodium dodecyl sulfate DNA extraction method was 98% reproducible when readable patterns were obtained. Reference strains from 43 of the 75 groups were cytotoxin positive, 28 groups were cytotoxin negative, and 4 groups included both toxigenic and nontoxigenic strains. Cytotoxicity of isolates with a particular REA type was always consistent with the toxicity of the reference strain for that type. REA typing was able to discriminate strain differences within types identified by the immunoblot (89 isolates), bacteriophage-bacteriocin (44 isolates), and ribotyping (23 isolates) methods. REA typing is a sensitive, discriminating, reproducible, and rapid method for differentiating C. difficile strains and is suitable for large-scale epidemiologic studies.

Characterization ofListeria strains from a foodborne listeriosis outbreak by rDNA gene restriction patterns compared to four other typing methods

The rDNA gene restriction patterns of 134 isolates of Listeria species were determined with pKK3535--a pBR322 derived plasmid containing an Escherichia coli rRNA operon--used as a probe following digestion of chromosomal DNA by EcoRI endonuclease. Nineteen reference and type strains representing all species and serotypes of Listeria showed 17 distinct ribotypes. One hundred and fifteen wild strains of Listeria monocytogenes were ribotyped and the results were compared to those of serotyping, phage typing, multilocus enzyme electrophoresis (MEE) and restriction endonuclease analysis (REA). Ninety-six Listeria monocytogenes serotype 4b wild strains displayed six distinct ribotypes (I-VI), 72% (69/96) of them clustering in two very close rDNA patterns (I and II) of eight and nine bands, respectively. The same 96 strains displayed six REA patterns and eight MEE electrotypes. Among the 96 Listeria monocytogenes 4b isolates, the 34 epidemic strains defined by phage typing and by epidemiological data all belonged to one ribotype (ribotype I) representing 56% of the strains belonging to this ribotype. These same 34 epidemic strains were also grouped by REA and MEE typing in a unique profile (REA-A) and MEE electrotype (ET 1). Twenty-two Listeria monocytogenes strains of serogroup 1/2 analyzed by rDNA typing showed nine distinct ribotypes. For the 96 Listeria monocytogenes 4b strains studied, the discriminatory index was highest for phage typing and for any combination including phage typing. Ribotyping appears to be a well reproducible molecular typing method and could be a useful complement to other typing methods for the epidemiological study of listeriosis.

Typing of Treponema hyodysenteriae by restriction endonuclease analysis

Restriction endonuclease analysis (REA) was used to type eight well-characterised strains of Treponema hyodysenteriae originating from the U.K., Canada and the U.S.A., and 16 isolates from cases of swine dysentery in Western Australia (W.A.). Several of the W.A. isolates were also serotyped by the method of Baum and Joens (1979), and the two typing techniques were compared. REA typing was more discriminatory than serotyping, being able to distinguish strains within serotypes. The new technique was neither more difficult nor more time-consuming to perform than serotyping. Within the 16 W.A. isolates, three different REA patterns were identified, with common patterns found on different farms. The eight overseas strains had seven different REA patterns, all of which could be distinguished from the patterns of the W.A. isolates.

Molecular epidemiology of Legionella species by restriction endonuclease and alloenzyme analysis.

As part of an ongoing investigation into nosocomial Legionella infections at Stanford University Medical Center (SUMC), we applied the technique of restriction endonuclease analysis (REA) to determine strain differences among three species, including Legionella pneumophila, Legionella dumoffii, and Legionella micdadei. A total of 26 human and environmental water isolates from SUMC were selected for REA and compared with control strains that were not epidemiologically linked to SUMC. REA results were compared with results of alloenzyme typing, typing by monoclonal antibodies, and plasmid fingerprinting in all but L. micdadei strains. REA and alloenzyme typing showed that SUMC patient isolates were derived from distinct strains of three species. L. pneumophila strains from SUMC patients were genotypically identical to those isolated from potable water. REA was especially useful in proving that SUMC L. dumoffii patient isolates were derived from a single strain and that patients may have been exposed to a common source(s). REA typing correlated well with alloenzyme typing. These methods complement serologic typing of L. pneumophila and provide discriminating capability between strains of other Legionella species such as L. dumoffii, for which serologic types have not been identified. In addition, REA typing is somewhat easier to perform than alloenzyme typing and can be done in clinical laboratories.