Restriction Endonuclease Analysis Group Research Articles

Editorial Commentary: Does Infection With Specific Clostridium difficile Strains or Clades Influence Clinical Outcome?

A number of DNA banding–based typing systems for classifying Clostridium difficile isolates have been devised including polymerase chain reaction (PCR) ribotyping, restriction endonuclease analysis (REA), and pulsed-field gel electrophoresis (PFGE) [1–4]. PCR ribotyping was developed in Wales and is commonly used in Europe, whereas REA and PFGE have been used more commonly in North America. In addition, toxinotyping, which is based on amplification of regions within the pathogenicity locus (PaLoc) for toxins A and B, has been used to describe “toxin variant” strains whose PaLoc genes differ from the standard or toxinotype “0” PaLoc to which most PCR ribotypes, REA groups, and PFGE types belong [5]. The aforementioned DNA-banding typing systems are invaluable for discriminating isolates from each other, but do not provide meaningful evolutionary analyses. Multilocus sequence typing (MLST), in which a selected number of housekeeping gene loci (7 in the case of C. difficile) are amplified and sequenced to yield sequence types (STs), can be used to group STs by evolutionary relationships into clades [6]. Members of a clade are considered to be derived from the same common ancestor and can be organized into evolutionary trees using phylogenetic or cladistic analyses. MLST classification of C. difficile clades agrees with previous classification based on DNA microarray comparative genomics and suggests that genotypes clustered by MLST may be an accurate proxy for whole-genome sequencing analysis [7]. STs correlate well with PCR ribotypes; however, as with toxinotyping, the vast majority of STs are clustered in a single clade, clade 1, with only 1 or 2 STs (ribotypes) in each of the remaining 4 clades (Table 1).

Relapse Versus Reinfection: Recurrent Clostridium difficile Infection Following Treatment With Fidaxomicin or Vancomycin

Our study sought to compare the strain types of Clostridium difficile causing initial and recurrent episodes of C. difficile infection (CDI) in adult patients with a first episode of CDI or 1 prior episode of CDI within the previous 90 days. Strains originated from patients who had been entered into two phase 3 randomized clinical trials of fidaxomicin versus vancomycin. Isolates of C. difficile from the initial and recurrent episodes within 28 (±2) days of cure of CDI were compared using restriction endonuclease analysis (REA) typing. Paired isolates were available from 90 of 194 (46%) patients with recurrent CDI. Patients with isolates available were significantly younger (P = .008) and more likely to be from Canadian sites (P = .0001), compared with patients without isolates. In 75 of 90 subjects (83.3%), the identical REA type strain was identified at recurrence and the initial episode (putative relapse). Early recurrences (0–14 days after treatment completion) were relapses in 86.7% and a new strain (reinfection) in 13.3%. Later recurrences (15–31 days after treatment) were relapses in 76.7% and reinfections in 23.3%. Mean time (± standard deviation) to recurrence was 12.2 (±6.4) days for relapses and 14.7 (±6.8) days for reinfections (P = .177). The most common BI/NAP1/027 group and the previous US epidemic REA group J/NAP2/001 had a significantly higher combined rate of recurrence with the same strain (relapse), compared with the other REA groups (39 of 42 [93%] vs 36 of 48 [75%], respectively; P = .023). We found a higher than historic rate of recurrent CDI caused by the same isolate as the original episode, a finding that may be related to the relatively short observation period in this study and the high frequency of isolation of epidemic strains, such as groups BI and J, for which relapse rates may be higher than for other REA groups. Caution in generalizing these observations is required, because the patients studied were younger and more likely to be from Canadian sites than were patients with recurrence who did not provide isolates.Clinical Trials Registration. NCT00314951 and NCT00468728.

Decreased Cure and Increased Recurrence Rates for Clostridium difficile Infection Caused by the Epidemic C. difficile BI Strain

An epidemic strain of Clostridium difficile designated by restriction endonuclease analysis (REA) as group BI has caused multiple outbreaks of severe C. difficile infection (CDI). The treatment response of patients infected with this strain is uncertain. Clostridium difficile isolates were collected from 2 phase 3 clinical trials comparing fidaxomicin to vancomycin and typed using REA. Clinical cure and recurrence outcomes were analyzed by strain type of the infecting organism, BI and non-BI, using both univariate and multivariate analyses. From 999 patients, 719 isolates were available for typing (356 fidaxomicin treated and 363 vancomycin treated). BI was the most common REA group (34% of isolates). Patients infected with BI had lower cure rates (86.6%; 214 of 247) than those infected with non-BI strains (94.3%; 445 of 472) (P < .001). The cure rate difference between the BI and non-BI patients was significant for both vancomycin (P = .02) and fidaxomicin (P = .007). BI patients had a recurrence rate of 27.4% (51 of 186), compared with a recurrence rate of 16.6% (66 of 397) in non-BI patients (P = .002). By multivariate analysis, BI infection was statistically significant as a risk factor for reduced cure (odds ratio [OR], 0.48; 95% confidence interval [CI], .27-.85; P = .030) and for increased recurrence (OR, 1.57; 95% CI, 1.01-2.45; P = .046). The clinical cure rate of patients infected with the epidemic BI C. difficile strain is lower than the cure rate of those infected with non-BI strains whether treated with fidaxomicin or vancomycin. Similarly, the CDI recurrence rate is increased in patients with the BI strain compared with patients with other C. difficile strains.

Comparative Susceptibilities to Fidaxomicin (OPT-80) of Isolates Collected at Baseline, Recurrence, and Failure from Patients in Two Phase III Trials of Fidaxomicin against Clostridium difficile Infection

A 10-day course of oral fidaxomicin (200 mg twice a day [b.i.d.]), a potent new macrocyclic drug, was compared to vancomycin (125 mg four times a day [q.i.d.]) in 1,164 adults (1,105 in the modified intent-to-treat [mITT] population) with Clostridium difficile infection in two phase III randomized, double-blind trials at sites in North America and 7 European countries. Of 1,105 mITT patients, 792 (71.7%), including 719/999 (72.0%) in the per-protocol (PP) population, provided a C. difficile strain at baseline, of whom 356 received fidaxomicin with 330 cures (92.7%) and 363 received vancomycin with 329 cures (90.6%). The susceptibilities (MIC(90)) of baseline isolates did not predict clinical cure, failure, or recurrence for fidaxomicin (MIC(90), 0.25 μg/ml for both; range, ≤ 0.007 to 1 μg/ml), but there was a one-dilution difference in the MIC(90) (but not the MIC(50)) for vancomycin (MIC(90), 2 μg/ml [range, 0.25 to 8 μg/ml] for cure and 4.0 μg/ml [range, 0.5 to 4 μg/ml] for failures). A total of 65 (7.9%) "rifaximin-resistant" (MIC > 256 μg/ml) strains were isolated in both treatment groups on enrollment, which increased to 25% for failures at the end of therapy. No resistance to either fidaxomicin or vancomycin developed during treatment in either of the phase III studies, although a single strain isolated from a cured patient had an elevated fidaxomicin MIC of 16 μg/ml at the time of recurrence. All isolates were susceptible to ≤ 4 μg/ml of metronidazole. When analyzed by restriction endonuclease analysis (REA) type, 247/719 (34.4%) isolates were BI group isolates, and the MICs were generally higher for all four drugs tested (MIC(90)s: fidaxomicin, 0.5; vancomycin, 2.0; metronidazole, 2.0; and rifaximin, >256 μg/ml) than for the other REA types. There was no correlation between the MIC of a baseline clinical isolate and clinical outcome. MIC(90)s were generally low for fidaxomicin and vancomycin, but BI isolates had higher MICs than other REA group isolates.

Comparison of Strain Typing Results for Clostridium difficile Isolates from North America

Accurate strain typing is critical for understanding the changing epidemiology of Clostridium difficile infections. We typed 350 isolates of toxigenic C. difficile from 2008 to 2009 from seven laboratories in the United States and Canada. Typing was performed by PCR-ribotyping, pulsed-field gel electrophoresis (PFGE), and restriction endonuclease analysis (REA) of whole-cell DNA. The Cepheid Xpert C. difficile test for presumptive identification of 027/NAP1/BI isolates was also tested directly on original stool samples. Of 350 isolates, 244 (70%) were known PCR ribotypes, 224 (68%) were 1 of 8 common REA groups, and 187 (54%) were known PFGE types. Eighty-four isolates typed as 027, NAP1, and BI, and 83 of these were identified as presumptive 027/NAP1/BI by Xpert C. difficile. Eight additional isolates were called presumptive 027/NAP1/BI by Xpert C. difficile, of which three were ribotype 027. Five PCR ribotypes contained multiple REA groups, and three North American pulsed-field (NAP) profiles contained both multiple REA groups and PCR ribotypes. There was modest concordance of results among the three methods for C. difficile strains, including the J strain (ribotype 001 and PFGE NAP2), the toxin A-negative 017 strain (PFGE NAP9 and REA type CF), the 078 animal strain (PFGE NAP7 and REA type BK), and type 106 (PFGE NAP11 and REA type DH). PCR-ribotyping, REA, and PFGE provide different but overlapping patterns of strain clustering. Unlike the other methods, the Xpert C. difficile 027/NAP1/BI assay gave results directly from stool specimens, required only 45 min to complete, but was limited to detection of a single strain type.

Killing kinetics of fidaxomicin and its major metabolite, OP-1118, against Clostridium difficile

The kinetics of bacterial killing for fidaxomicin and its major metabolite, OP-1118, were investigated against Clostridium difficile strains, including two clinical strains belonging to the restriction endonuclease analysis group BI (ORG 1687 and 1698), the ATCC 43255 strain and two laboratory-derived mutant strains with decreased susceptibility to fidaxomicin (ORG 919 and 1620). Both fidaxomicin and OP-1118 demonstrated time-dependent killing of C. difficile strains. Fidaxomicin (at 4× MIC) reduced bacterial counts of the ATCC 43255 strain, clinical strain ORG 1687 and the two laboratory-generated mutant strains by ≥3 logs within 48 h of exposure. The other BI strain, ORG 1698, was tested at 2× MIC fidaxomicin with bacterial counts decreasing 1 log in 48 h. Exposure to OP-1118 (at 4× MIC) also resulted in a ≥3 log drop in c.f.u. counts for the ATCC 43255 strain, the clinical BI strain ORG 1687 and the mutant strain ORG 919. Higher concentrations of OP-1118 (32× MIC) were required for a 3 log reduction in c.f.u. counts for the other BI strain, ORG 1698. In summary, the results indicate that both fidaxomicin and its major metabolite, OP-1118, are bactericidal against C. difficile strains, including the hypervirulent restriction endonuclease analysis group BI strains, at concentrations that are many fold below the detected faecal concentrations of these compounds after oral administration of fidaxomicin.

Update on Clostridium difficile infection

This review summarizes the most recent epidemiological data and advances in research into the pathogenesis, diagnosis and treatment of Clostridium difficile infection (CDI). The epidemiology of CDI has changed with the emergence of hypervirulent strains. CDI rates have increased in the community, in children and in patients with inflammatory bowel disease. Although the North American pulsed-field gel electrophoresis type 1, restriction endonuclease analysis group BI, PCR ribotype 027 (NAP1/BI/027) strain remains prevalent in North America, surveillance suggests that it is decreasing in Europe. A similar strain, PCR ribotype 078, is emerging which is associated with community-associated CDI and has been isolated in animals and food products. The Society for Healthcare Epidemiology of America and the Infectious Diseases Society of America have published new guidelines on the epidemiology, diagnosis, treatment, infection control and environmental management of C. difficile. Several novel therapies for CDI are at different stages of development. There have been promising trial results with fidaxomicin, a novel antibiotic for the treatment of CDI and monoclonal antibodies against toxins A and B, which have been shown to significantly reduce CDI recurrence rates. Major advances have been made in our understanding of the spread and pathogenesis of C. difficile and new treatment options are becoming available.

Decrease of hypervirulent Clostridium difficile PCR ribotype 027 in the Netherlands

After the first outbreaks of Clostridium difficile PCR ribotype 027 (North American pulsed-field type 1, restriction endonuclease analysis group BI) in the Netherlands in 2005, a national surveillance programme for C. difficile infection (CDI) was started. Furthermore, national guidelines were developed to rapidly recognise type 027 infections and prevent further spread. The mean incidence of CDI measured in 14 hospitals remained stable throughout the years: an incidence of 18 per 10,000 admissions was seen in 2007 and 2008. Between April 2005 and June 2009 a total of 2,788 samples were available for PCR ribotyping. A decrease was seen in the number and incidence of type 027 after the second half of 2006. In the first half of 2009, the percentage of type 027 isolates among all CDI decreased to 3.0%, whereas type 001 increased to 27.5%. Type 014 was present in 9.3% of the isolates and C. difficile type 078 slightly increased to 9.1%. We conclude that currently there is a significant decrease in type 027-associated CDI in the Netherlands.

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.

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.

Detection of virulence genes of Clostridium difficile by multiplex PCR

Clostridium difficile strains belonging to the PCR ribotype 027, pulse-field gel electrophoresis (PFGE) type NAP1, toxinotype III and restriction endonuclease analysis group BI harbouring mutations in the tcdC gene and possessing binary toxin components A and B have been described to cause epidemics with increased morbidity and mortality. In the present study we developed a conventional multiplex PCR designed to detect selected virulence associated markers of the hypervirulent C. difficile PCR ribotype 027. The multiplex PCR assay detected the major toxins A and B, binary toxin components A and B as well as a possible deletion in the tcdC gene: a characteristic pattern of amplification products for the PCR ribotype 027 strains was detected. This rather simple method was specific for the screening of this hypervirulent C. difficile strain. The correlation between the multiplex PCR and PCR ribotyping methods was excellent. The sensitivity and specificity were 100% in our epidemiological situation. In conclusion, this multiplex PCR was found useful in the preliminary screening for the hypervirulent C. difficile PCR ribotype 027.

Clostridium difficile Infection Caused by the Epidemic BI/NAP1/027 Strain

Rates and severity of Clostridium difficile infection (CDI) in hospitals in North America and Europe have increased since 2000 and correlate with dissemination of an epidemic strain characterized by higher than usual toxin A and B production, the presence of a third toxin, binary toxin, and high-level resistance to fluoroquinolone antibiotics. The strain, which is restriction endonuclease analysis group BI, pulse-field gel electrophoresis type NAP1, and polymerase chain reaction ribotype 027, is designated BI/NAP1/027. How this strain has become so widely distributed geographically and produces such severe CDI is the subject of active investigation. The deletion at position 117 of the tcdC gene, a repressor of toxin A and B production, is one possible contributor to increased levels of the toxins. The role of binary toxin is unknown. Recent isolates of BI/NAP1/027 were found to be resistant to fluoroquinolones, which is likely to contribute to the dissemination of this strain. Other virulence factors such as increased sporulation and surface layer protein adherence are also under investigation. Infections caused by this organism are particularly frequent among elderly hospitalized patients, in whom the attributable 30-day mortality is greater than 5%. Major risk factors for BI/NAP1/027 infection include advanced age, hospitalization, and exposure to specific antimicrobials, especially fluoroquinolones and cephalosporins. When CDI is severe, vancomycin treatment is more effective than metronidazole; for mild disease either agent can be used. Control of hospital outbreaks caused by BI/NAP1/027 is difficult but possible through a combination of barrier precautions, environmental cleaning, and antimicrobial stewardship.

Editorial Commentary:Decreased Effectiveness of Metronidazole for the Treatment ofClostridium difficileInfection?

Since the emergence of a new virulent strain of Clostridium difficile characterized as toxinotype III, North American PFGE type 1 (NAP1), restriction endonuclease analysis group type BI, and PCR ribotype 027 (type 027) multiple outbreaks have been reported in North America and Europe [1, 2]. The first reports were from Canada, where the province of Quebec was the earliest and most severely affected [3]. In the United States, at least 38 states have been affected by the new emerging strain [4]. Outbreaks in the United Kingdom and The Netherlands have been followed by a rapid spread of the strain throughout Europe, now including 18 countries [5]. A worrisome new development is outbreaks in Europe attributable to clindamycin-resistant NAP1/027 strains (MIC, >256 mg/L) that harbor the ermB gene [6]. Clindamycin has been considered to be a protective antibiotic for the development of C. difficile-associated disease (CDAD) attributable to NAP1/027, but resistance to this agent increases the risk of C. difficile in-

Antimicrobial‐Associated Risk Factors forClostridium difficileInfection

Antimicrobial therapy plays a central role in the pathogenesis of Clostridium difficile infection (CDI), presumably through disruption of indigenous intestinal microflora, thereby allowing C. difficile to grow and produce toxin. Investigations involving animal models and studies performed in vitro suggest that inhibitory activity against C. difficile and differences in the propensity to stimulate toxin production may also influence the likelihood that particular drugs may cause CDI. Although nearly all antimicrobial classes have been associated with CDI, clindamycin, third-generation cephalosporins, and penicillins have traditionally been considered to harbor the greatest risk. Recent studies have also implicated fluoroquinolones as high-risk agents, a finding that is most likely to be related in part to increasing fluoroquinolone resistance among epidemic strains (i.e., restriction-endonuclease analysis group BI/North American PFGE type 1 strains) and some nonepidemic strains of C. difficile. Restrictions in the use of clindamycin and third-generation cephalosporins have been associated with reductions in CDI. Because use of any antimicrobial has the potential to induce the onset of CDI and disease caused by other health care-associated pathogens, antimicrobial stewardship programs that promote judicious use of antimicrobials are encouraged in concert with environmental and infection control-related efforts.

C difficile—C’est Difficile?

C difficile—C’est Difficile?

An Epidemic, Toxin Gene–Variant Strain ofClostridium difficile

Recent reports suggest that the rate and severity of Clostridium difficile-associated disease in the United States are increasing and that the increase may be associated with the emergence of a new strain of C. difficile with increased virulence, resistance, or both. A total of 187 C. difficile isolates were collected from eight health care facilities in six states (Georgia, Illinois, Maine, New Jersey, Oregon, and Pennsylvania) in which outbreaks of C. difficile-associated disease had occurred between 2000 and 2003. The isolates were characterized by restriction-endonuclease analysis (REA), pulsed-field gel electrophoresis (PFGE), and toxinotyping, and the results were compared with those from a database of more than 6000 isolates obtained before 2001. The polymerase chain reaction was used to detect the recently described binary toxin CDT and a deletion in the pathogenicity locus gene, tcdC, that might result in increased production of toxins A and B. Isolates that belonged to one REA group (BI) and had the same PFGE type (NAP1) were identified in specimens collected from patients at all eight facilities and accounted for at least half of the isolates from five facilities. REA group BI, which was first identified in 1984, was uncommon among isolates from the historic database (14 cases). Both historic and current (obtained since 2001) BI/NAP1 isolates were of toxinotype III, were positive for the binary toxin CDT, and contained an 18-bp tcdC deletion. Resistance to gatifloxacin and moxifloxacin was more common in current BI/NAP1 isolates than in non-BI/NAP1 isolates (100 percent vs. 42 percent, P<0.001), whereas the rate of resistance to clindamycin was the same in the two groups (79 percent). All of the current but none of the historic BI/NAP1 isolates were resistant to gatifloxacin and moxifloxacin (P<0.001). A previously uncommon strain of C. difficile with variations in toxin genes has become more resistant to fluoroquinolones and has emerged as a cause of geographically dispersed outbreaks of C. difficile-associated disease.

A Hospital Outbreak of Clostridium difficile Disease Associated with Isolates Carrying Binary Toxin Genes

The binary toxin genes cdt and cdtB have been detected in approximately 5% of Clostridium difficile strains. Severe C. difficile disease (CDD) may be associated with strains that carry the binary toxin genes. From April 2001 through March 2002, 8 severe and 41 nonsevere cases of nosocomial CDD were studied. Severe cases of CDD were defined by the presence of >or=2 of the following criteria: (1) abdominal pain, (2) a white blood cell count of >20,000 or <1500 cells/mm(3), and (3) ileus or bowel wall thickening with ascites. Underlying disease was assessed by 2 methods: a modified Horn score and the presence of comorbid conditions. The presence of cdtA, cdtB, and the toxin A and toxin B genes was determined, and molecular subtyping was performed. All strains were positive for the toxin A and B genes, and 65.3% of the strains carried the cdtA and cdtB genes. Strains that carried the binary toxin genes accounted for 87.5% of the cases of severe CDD and 61.0% of the nonsevere cases (P=.23). Severity of CDD was not associated with either severe underlying disease or comorbid conditions. The strains that caused severe CDD belonged to 4 protein profile groups and >or=3 restriction endonuclease analysis (REA) groups. All (i.e., 5 of 5) strains in REA group BI, compared with none (i.e., 0 of 7) of the strains in REA group J carried the binary toxin genes (P=.001). Strains that belonged to REA groups BK and BR also carried the binary toxin genes. The binary toxin genes were present in nearly two-thirds of the C. difficile strains, and they were correlated with the REA group. Severity of CDD was not closely associated with a specific clone or underlying disease, but it may be associated with the presence of the binary toxin genes. Larger studies are needed to discern whether a true association exists and whether the binary toxin alters the pathogenicity of the C. difficile strain.

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.

Toxin gene analysis of a variant strain of Clostridium difficile that causes human clinical disease.

A toxin variant strain of Clostridium difficile was isolated from two patients with C. difficile-associated disease (CDAD), one of whom died from extensive pseudomembranous colitis. This strain, identified by restriction endonuclease analysis (REA) as type CF2, was not detected by an immunoassay for C. difficile toxin A. Culture supernatants of CF2 failed to elicit significant enterotoxic activity in the rabbit ileal loop assay but did produce atypical cytopathic effects in cell culture assay. Southern hybridization, PCR amplification, and DNA sequence analyses were performed on the toxin A (tcdA) and toxin B (tcdB) genes of type CF2 isolate 5340. Type CF2 5340 tcdA exhibited a 1,821-bp truncation, due to three deletions in the 3' end of the gene, and a point mutation in the 5' end of the gene, resulting in a premature stop codon at tcdA position 139. Type CF2 5340 tcdB exhibited multiple nucleotide base substitutions in the 5' end of the gene compared to tcdB of the standard toxigenic strain VPI 10463. Type CF2 5340 toxin gene nucleotide sequences and deduced amino acid sequences showed a strong resemblance to those of the previously described variant C. difficile strain 1470, a strain reported to have reduced pathogenicity and no association with clinical illness in humans. REA of strain 1470 identified this strain as a distinct type (CF1) within the same REA group as the closely related type CF2. A review of our clinical-isolate collection identified five additional patients infected with type CF2, three of whom had documented CDAD. PCR amplification of the 3' end of tcdA demonstrated identical 1. 8-kb deletions in all seven type CF2 isolates. REA type CF2 is a toxin variant strain of C. difficile that retains the ability to cause disease in humans but is not detected in clinical immunoassays for toxin A.

Analysis of plasmid and chromosomal DNA of multidrug-resistant Salmonella enterica serovar typhi from Asia.

Molecular analysis of chromosomal DNA from 193 multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates from 1990 to 1995 from Pakistan, Kuwait, Malaysia, Bangladesh, and India produced a total of five major different pulsed-field gel electrophoresis (PFGE) patterns. Even within a particular country MDR S. enterica serovar Typhi DNA was found to be in different PFGE groups. Similar self-transferable 98-MDa plasmids belonging to either incompatibility group incHI1 or incHI1/FIIA were implicated in the MDR phenotype in S. enterica serovar Typhi isolates from all the locations except Quetta, Pakistan, where the majority were of incFIA. A total of five different PFGE genotypes with six different plasmids, based on incompatibility and restriction endonuclease analysis groups, were found among these MDR S. enterica serovar Typhi isolates.