Isolation and molecular characterization of Clostridium difficile strains from patients and the hospital environment in Belarus

Toxigenic Clostridium difficile is the most common etiologic agent of hospital-acquired diarrhea in developed countries. The role of this pathogen in nosocomial diarrhea in Eastern Europe has not been clearly established. The goal of this study was to determine the prevalence of C. difficile in patients and the hospital environment in Belarus and to characterize these isolates as to the presence of toxin genes and their molecular type. C. difficile was isolated from 9 of 509 (1.8%) patients analyzed and recovered from 28 of 1,300 (2. 1%) environmental sites cultured. A multiplex PCR assay was used to analyze the pathogenicity locus (PaLoc) of all isolates, and strain identity was determined by an arbitrarily primed PCR (AP-PCR). The targeted sequences for all the genes in the PaLoc were amplified in all C. difficile strains examined. A predominantly homogeneous group of strains was found among these isolates, with five major AP-PCR groups being identified. Eighty-three percent of environmental isolates were classified into two groups, while patient isolates grouped into three AP-PCR types, two of which were also found in the hospital environment. Although no data on the role of C. difficile infection or epidemiology of C. difficile-associated diarrhea (CDAD) in this country exist, the isolation of toxigenic C. difficile from the hospital environment suggests that this pathogen may be responsible for cases of diarrhea of undiagnosed origin and validates our effort to further investigate the significance of CDAD in Eastern Europe.     

Epidemiology of Clostridium difficile infection in a large teaching hospital in Singapore

AIMS: We undertook this study to define the incidence of toxigenic Clostridium difficile in our hospital and to characterise the isolates. METHODS: All unformed stool was tested for the presence of Toxin A (TcdA) and Toxin B (TcdB), and cultured for C. difficile. Culture filtrates were also tested for TcdA and TcdB. Detection of tcdA and tcdB genes was carried out for A(-)B(+) strains by polymerase chain reaction (PCR).The minimum inhibitory concentrations (MICs) of metronidazole, vancomycin and clindamycin for all isolates were tested using the Etest. PCR ribotyping was carried out on all isolates. RESULTS: The incidence of Clostridium difficile associated disease (CDAD) was 3.2 cases per 1000 admissions or discharges and 53.8 cases per 100 000 patient days. Most cases occurred in renal and haematology patients. CDAD was more common in patients aged over 50 years and of male gender. The Indian population was under-represented. Fourteen (11.8%) isolates were A(-)B(+). All strains were susceptible to metronidazole but one strain showed intermediate resistance to vancomycin. Only 12.8% of the isolates were susceptible to clindamycin. Thirty-five isolates had PCR ribotype A, of which 29 (83%) had a clindamycin MIC >256 mg/L. Thirty-three had PCR ribotype B, of which only one (3%) had a clindamycin MIC >256 mg/L. The 14 A(-)B(+) strains were all PCR ribotype C, and had a range of MICs for clindamycin from 2 to >256 mg/L. CONCLUSIONS: The incidence of CDAD in our hospital is relatively low. Isolates remain susceptible to metronidazole and vancomycin.     

Clinical characteristics of Clostridium difficile infection in hospitalized patients with antibiotic-associated diarrhea in a university hospital in China

The purpose of this study was to identify clinical characteristics of Clostridium difficile infection (CDI) in patients with antibiotic-associated diarrhea (AAD). A prospective study was conducted among patients hospitalized in Fudan University Hospital Huashan from August 1, 2012 to July 31, 2013. Toxigenic C. difficile isolates were characterized by PCR ribotyping and multilocus sequence typing. AAD developed in 1.0 % (206/20437) of the antibiotic-treated hospitalized patients and toxigenic C. difficile was isolated from 30.6 % (63/206) of patients with AAD. The frequency of AAD was highest in the intensive care unit (10.7 %); however the proportion of CDI in AAD was highest in the Geriatric Unit (38 %). AAD ranged in severity from mild to moderate. One case with pseudomembranous colitis was identified. Use of carbapenems was found to significantly increase the risk of CDI (OR, 2.31; 95 % CI, 1.22–4.38; p?=?0.011). Patient demographics, presumed risk factors, clinical manifestations and laboratory findings revealed no significant difference between patients with CDI and non-C. difficile AAD. Over 90 % of the patients with CDI or non-C. difficile AAD were cured. Two patients had CDI recurrence. Ribotype H was the dominant (18.8 %) genotype, followed by ribotype 012 and ribotype 017. C. difficile plays a significant role in AAD in our setting in China. Because the severity of diarrhea ranges from mild to moderate, it is difficult for Chinese clinicians to identify CDI from AAD patients, therefore CDI should be included in the routine differential diagnoses for hospitalized patients presenting with AAD.     

Epidemiology and clinical characteristics of Clostridium difficile infection in a Korean tertiary hospital

In order to investigate the incidence, clinical and microbiologic characteristics of Clostridium difficile infection (CDI) in Korea, a prospective observational study was performed. From September 2008 through January 2010, all patients whose stool was tested for toxin assay A&B and/or C. difficile culture were studied for clinical characteristics. Toxin types of the isolates from stool were tested. The mean incidence of CDI per 100,000 patient-days was 71.6 by month (range, 52.5-114.0), and the ratio of CDI to antibiotic-associated diarrhea was 0.23. Among 200 CDI patients, 37.5% (75/200) was severe CDI based on severity score. Clinical outcome of 189 CDI was as followed; 25.9% (49/189) improved without treatment, 84.3% (118/140) achieved clinical cure and attributed mortality was 0.7% (1/140) with the treatment. Recurrence rate was 21.4% (30/140) and cure without recurrence was 66.4% (93/140). The most common type of toxin was toxin A-positive/toxin B-positive strain (77.5%), toxin A-negative/toxin B-positive strains or binary toxin-producing strains comprised 15.4% or 7.1%, respectively. In conclusion, the incidence of CDI in Korea is a little higher than other reports during the non-epidemic setting. We expect that the change of epidemiology and clinical severity in CDI can be evaluated based on these results.     

Clinical features of Clostridium difficile infection and molecular characterization of the isolated strains in a cohort of Danish hospitalized patients

The purpose of this study was to compare clinical features of Clostridium difficile infection (CDI) to toxin gene profiles of the strains isolated from Danish hospitalized patients. C. difficile isolates were characterized by PCR based molecular typing methods including toxin gene profiling and analysis of deletions and truncating mutations in the toxin regulating gene tcdC. Clinical features were obtained by questionnaire. Thirty percent of the CDI cases were classified as community-acquired. Infection by C. difficile with genes encoding both toxin A, toxin B and the binary toxin was significantly associated with hospital-acquired/healthcare-associated CDI compared to community-acquired CDI. Significantly higher leukocyte counts and more severe clinical manifestations were observed in patients infected by C. difficile containing genes also encoding the binary toxin together with toxin A and B compared to patients infected by C. difficile harbouring only toxin A and B. In conclusion, infection by C. difficile harbouring genes encoding both toxin A, toxin B and the binary toxin were associated with hospital acquisition, higher leukocyte counts and severe clinical disease.     

Prevalence and genetic characterization of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France

Toxin A variant strains (toxin A-negative, toxin B-positive strains) of Clostridium difficile have been reported to be responsible for diarrhea or pseudomembranous colitis in humans. These strains lack parts of the repeating sequences of the toxin A gene (tcdA) and are toxin A negative by commercial enzyme immunoassays (EIA). Here, we report the prevalence of the toxin A variant strains in 334 patients with C. difficile-associated diarrhea in France. The repeating segment of the tcdA gene (1,200 bp) was amplified by PCR using the primers NK9 and NK11 (H. Kato et al., J. Clin. Microbiol. 36:2178-2182, 1998). In the case of amplified fragments of unexpected size, the entire tcdA gene was studied by PCRs A1, A2, and A3 (Rupnik et al., J. Clin. Microbiol. 36:2240-2247, 1998), and strains were characterized by serotyping, pulsed-field gel electrophoresis and PCR ribotyping. By PCR with primers NK9 and NK11, C. difficile variant strains were detected in 2.7% of patients. Several variant types were found. A deletion of approximately 1,700 bp was observed in six strains from five patients. These strains belonged to serotype F and were characterized by the same pulsotype and the same PCR ribotype. They were toxin A negative by EIA and exhibited an atypical cytopathic effect on MRC-5 cells. Two other tcdA variant types that exhibited a positive result for toxin A by EIA were identified: one from serotype H with a longer amplified fragment (insertion of 200 bp) and one with a deletion of 600 bp. Diagnosis of C. difficile-associated diseases would have been missed in five patients (1.5%) by laboratories that screen the stools only for the presence of toxin A. This result underlines the need for testing stool by the cytotoxicity assay in patients with a high suspicion of C. difficile-associated diarrhea but a negative immunoassay for toxin A.     

Clonal dissemination of a toxin-A-negative/toxin-B-positive Clostridium difficile strain from patients with antibiotic-associated diarrhea in Poland

Objective To determine the incidence of toxin-A-negative/toxin-B-positive Clostridium difficile strains and their genetic relatedness in the feces of patients suffering from antibiotic-associated diarrhea (AAD) in Polish hospitals.
Methods C. difficile strains were cultured from patients' stool samples. The present study characterises these strains with respect to their cytopathogenicity on McCoy cells and the absence of toxin A despite a functional toxin B as determined with commercial test kits (Culturette Brand Toxin CD-TCD toxin A test and C. difficile Tox A/B test). In addition, PCR using different primer pairs aiming at non-repeating or repeating regions of the toxin A and B genes were used to confirm the findings. All toxin A^–B⁺ strains were genetically characterised by random amplification of polymorphic DNA (RAPD) analysis, PCR ribotyping and, in part, pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments.
Results We here present the presence of 17 toxin A^–B⁺ strains among 159 C. difficile strains (11%) isolated from fecal samples from 413 patients with antibiotic-associated diarrhea. All 17 strains possessed the toxin B gene, demonstrated a cytopathogenic effect on the McCoy cells, and were positive in the Tox A/B test. Molecular typing of these 17 C. difficile strains revealed that 7 of 17 (41%) toxin A^–/B⁺ C. difficile strains could not be discriminated. It appeared that these strains had a genotype that could not be distinguished from that of a Japanese control strain.
Conclusion Our observations imply that a particular genotype of toxin A^–B⁺ C. difficile has spread extensively, not only in Poland but possibly even worldwide.     

Experimental reproduction of neonatal diarrhea in young gnotobiotic hares simultaneously associated with Clostridium difficile and other Clostridium strains.

Clostridium difficile, C. perfringens, and C. tertium are very often present simultaneously in the feces of conventional diarrheic young hares, whereas these three bacterial species are rarely encountered and never present simultaneously in the feces of healthy young hares. When a strain of each of the three bacterial species was monoassociated with axenic young hares, the appearance of pathological disorders was only observed in animals monoassociated with C. difficile, when the number of C. difficile exceeded 10(8) per g of fresh feces. When a strain of C. perfringens or a strain of C. tertium, or both, was associated with C. difficile, diarrhea and death occurred more rapidly than in hares monoassociated with C. difficile. C. difficile and C. perfringens became established more rapidly when disassociated than when monoassociated with axenic hares. The association of C. perfringens and C. tertium with axenic hares did not bring about any pathological disorders. It may be concluded that C. difficile is the causal agent of neonatal diarrhea in conventional and gnotobiotic young hares and that other strains of Clostridium enhance its pathogenic effect. C difficile alone or associated with C. perfringens or C. tertium does not play any pathogenic role in young rats, mice, or rabbits.     

Electron microscopic investigation of lysogeny of Clostridium difficile strains isolated from antibiotic-associated diarrhea cases and from healthy carriers

An electron microscopic investigation was performed on 28 Clostridium difficile strains isolated from 15 antibiotic-associated diarrhea cases and from 13 healthy infants. Through the use of supernatants of the cultures induced by mitomycin C (1 or 3 micrograms/ml), 18 of the 28 C. difficile strains proved to harbor phage particles with a different morphology and size. Most of the phages revealed belonged in phage group B1 with a non-contractile tail. Seven of the 11 toxin-producing, lysogenic C. difficile strains carried a defective phage structure (120 nm-long tail with an incomplete head capsule) alone or together with other normal phages. With different C. difficile strains as indicator, plaque formation could not be detected in any of the lysates.     

Electrophoretic characterization of Clostridium difficile strains isolated from antibiotic-associated colitis and other conditions.

Clostridium difficile has been recognized as the cause of antibiotic-associated pseudomembranous colitis and of less severe diarrheal diseases associated with the use of antimicrobial agents. However, healthy carriers of this microorganism have been found, particularly healthy neonates and small children. Various typing systems have been used to clarify the epidemiology of C. difficile. We used the electrophoretic patterns of EDTA-extracted proteins to characterize C. difficile strains from various sources. Altogether, 110 strains were studied, including 2 reference strains, and 21 different protein profiles were obtained. However, two patterns were the most common: the group 2 pattern, characterized by a major 35-kilodalton polypeptide band, and the group 5 pattern, identified by principal bands of 37 and 56 kilodaltons. The group 2 pattern was characteristic of strains isolated during hospital outbreaks and from sporadic cases of pseudomembranous colitis and antibiotic-associated diarrhea. The group 5 pattern was obtained only from isolates from healthy neonates and children. A correlation between electrophoretic characteristics and virulence can be hypothesized, namely that group 2 strains are more prone to induce diseases and cause outbreaks. It is noteworthy that strains isolated from children with diarrhea of unknown etiology, not related to antibiotic use, belong to the "virulent" group 2; strains from leukemic patients showed a variety of different patterns, and only two belong to group 2. This characterization can be used to aid studies on the virulence and clinical significance of C. difficile.     

PCR ribotyping and arbitrarily primed PCR for typing strains of Clostridium difficile from a Polish maternity hospital.

Detection of the source of Clostridium difficile strains is of importance for the control of the nosocomial spread of this microorganism. For this purpose, vaginal and rectal swabs from 183 mothers, duplicate fecal samples (taken on days 1 and 4 after birth) from 183 neonates, and 94 environmental samples were cultured for C. difficile. The microorganism was never detected in the meconium obtained on day 1 after birth. On the other hand, an incidence of 17% C. difficile positivity was noted in the fecal samples obtained on day 4 after birth. Forty-two percent of the 31 colonized neonates had been delivered with complications. The bacteria were never encountered in the rectal swabs of the mothers, and C. difficile was identified in only one vaginal swab. In contrast, 13% of the environmental samples were positive for C. difficile. No major difference was encountered between patient and environmental isolates with respect to toxigenicity (58 to 65% toxigenic isolates). All strains were subsequently typed by PCR amplification of the 16S-23S ribosomal intergenic spacer regions and by arbitrarily primed PCR (AP-PCR) with different primers and combinations thereof. All environmental isolates and 11 of 31 neonatal strains were of a single type. The vaginal strain was unique, and among the maternity ward- and neonate-related isolates, only two additional AP-PCR types were identified. When a collection of C. difficile strains from patients hospitalized in other institutions and suffering from antibiotic-associated diarrhea or pseudomembranous colitis was analyzed in a similar manner, it appeared that the strain from the maternity ward was unique.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of Clostridium difficile from patients and the environment of hospital wards.

Rectal swabs from 122 patients and 497 environmental swabs from several wards were examined for the presence of Clostridium difficile in order to assess the role of the environment in the spread of this organism. Clostridium difficile was isolated from 6/27 (22.2%) oncology patients and from 8/163 (4.9%) environmental specimens obtained from the oncology unit. Items found positive for C difficile were those subjected to faecal contamination such as commode chairs, bed pans, dust pans, discard bins, the sluice and a disposable bed pan machine. Fourteen of 51 (27.4%) asymptomatic neonates yielded mostly toxigenic C difficile in their stools during their first week of life. Five of 156 (3.2%) specimens taken from inanimate objects in the environment of the neonatal units were positive for C difficile. The organism was also isolated from the hands of a nurse. Similar antibiogram patterns were demonstrated in the strains obtained from the patients and their environment indicating the possible occurrence of cross infection. These results indicate that environmental contamination is important in the spread of C difficile in hospitalised patients and the implementation of isolation procedures may limit that spread.     

Relative frequency of Clostridium difficile in patients with diarrheal disease.

We have studied 161 patients with diarrheal disease to determine the frequency with which Clostridium difficile occurs in such patients. C. difficile or its toxin or both were detected in stools from 19 patients (11.9%), 17 of whom had previously received antimicrobial agents. Enteric pathogens other than C. difficile were recovered less frequently, with Salmonella sp., Giardia lamblia, and Campylobacter fetus being recovered from 4.1, 2.5, and 1.3%, respectively, of the patients studied. These data suggest that C. difficile may be frequently encountered in specimens obtained from patients with diarrhea who receive antibiotics and may play a role in diarrheal disease in such patients.     

Clostridium difficile and its cytotoxin in feces of patients with antimicrobial agent-associated diarrhea and miscellaneous conditions.

Fecal specimens from 223 subjects were evaluated for the presence of Clostridium difficile by use of a selective medium developed in our laboratory and for the presence of C. difficile cytotoxin. C. difficile and cytotoxin were detected in 89 and 83%, respectively, of patients with antimicrobial agent-associated pseudomembranous colitis (PMC). In patients in whom PMC was not documented, C. difficile and cytotoxin were present in only 37 and 21%, respectively. C. difficile and cytotoxin were also recovered from the feces of 6 and 3, respectively, of 13 antimicrobial recipients who did not have diarrhea. Although C. difficile appears to be a major cause of PMC, it is not responsible for at least some two-thirds of cases of antimicrobial agent-associated diarrhea in which PMC is not documented. Neither the recovery of C. difficile nor the detection of its cytotoxin should be considered diagnostic for C. difficile-induced disease.     

Molecular epidemiology of hospital-associated and community-acquired Clostridium difficile infection in a Swedish county

All episodes of Clostridium difficile associated diarrhea (CDAD) diagnosed in a defined population of 274,000 including one tertiary and two primary hospitals and their catchment areas were studied during 12 months. The annual CDAD incidence in the county was 97 primary episodes per 100,000, and 78% of all episodes were classified as hospital associated with a mean incidence of 5.3 (range, 1.4 to 6.5) primary episodes per 1,000 admissions. The incidence among hospitalized individuals was 1,300-fold higher than that in the community (33,700 versus 25 primary episodes per 100,000 persons per year), reflecting a 37-fold difference in antibiotic consumption (477 versus 13 defined daily doses [DDD]/1,000 persons/day) and other risk factors. Three tertiary hospital wards with the highest incidence (13 to 36 per 1,000) had CDAD patients of high age (median age of 80 years versus 70 years for other wards, P < 0.001), long hospital stay (up to 25 days versus 4 days), or a high antibiotic consumption rate (up to 2,427 versus 421 DDD/1,000 bed days). PCR ribotyping of C. difficile isolates available from 330 of 372 CDAD episodes indicated nosocomial acquisition of the strain in 17 to 27% of hospital-associated cases, depending on the time interval between index and secondary cases allowed (2 months or up to 12 months), and only 10% of recurrences were due to a new strain of C. difficile (apparent reinfection). In other words, most primary and recurring episodes were apparently caused by the patient's endogenous strain rather than by one of hospital origin. Typing also indicated that a majority of C. difficile strains belonged to international serotypes, and the distribution of types was similar within and outside hospitals and in primary and relapsing CDAD. However, type SE17 was an exception, comprising 22% of hospital isolates compared to 6% of community isolates (P = 0.008) and causing many minor clusters and a silent nosocomial outbreak including 36 to 44% of the CDAD episodes in the three high-incidence wards.