Typing of Clostridium difficile

Clostridium difficile is primarily recognised as a nosocomially acquired pathogen manifesting in gastrointestinal disease subsequent to the patient receiving broad-spectrum antibiotics. Infection can be sporadic, but outbreaks commonly occur within a ward or hospital as a result of cross-infection. Since the 1980s, the epidemiology of C. difficile disease has been studied by the application of many different typing or fingerprinting methods; these, and the lessons learned, are reviewed herein.     

Laboratory diagnosis of Clostridium difficile disease

The laboratory diagnosis of Clostridium difficile -associated disease (CDAD) is based on culture and toxin detection in fecal specimens. Culture is performed on a commercially available selective media. C. difficile colony morphology is typical when viewed under a dissecting microscope. Definitive identification is best obtained by gas liquid chromatography. Culture is very sensitive but, when used alone without toxin testing, it leads to low specificity and misdiagnosis of CDAD when high rates of asymptomatic carriage exist. Toxin detection by a tissue culture cytotoxin assay followed by neutralisation with specific antiserum is often considered the standard. However, this approach lacks sensitivity and has not detected up to 30% of patients with confirmed CDAD. Multiple enzyme immunoassays (EIAs) have been introduced by various manufacturers for the detection of toxin A alone or for both toxins A and B. Some of these are designed to give results in less than 1 h. Comparative studies of EIA kits reported that the sensitivity and specificity are slightly lower than cytotoxin assays. Toxigenic culture tests C. difficile isolates for toxin production: colonies isolated on selective media are tested for in-vitro toxin production either by a cytotoxicity assay or by direct EIA. It has higher sensitivity than the cytotoxicity assay and equivalent specificity. In the routine laboratory, culture and toxin detection should be performed on every specimen and, in culture-positive and fecal toxin-negative cases, toxigenic cultures should be performed on isolated colonies.     

An immunochemical method for fingerprinting Clostridium difficile

The use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis in association with electrophoretic transfer of proteins to nitrocellulose and subsequent probing with antisera appears useful as a method for fingerprinting Clostridium difficile. Thorough testing of the stability of the antigenic nature of isolates of the organism during subculture and antigen preparation has shown it to be remarkably stable both in vitro and in vivo. Minor differences in the method of antigen extraction do not markedly alter the immunoblot patterns produced. It has also been demonstrated that an individual may harbour more than one strain of the organism at any one time. Results show the possible usefulness of this technique in studying the epidemiology of diarrhoeal disease known to be associated with C. difficile. It is suggested that for any serious study several colonies should be subcultured from the primary isolation plate.     

Genomic and phenotypic diversity of Clostridium difficile during long-term sequential recurrences of infection

Infection with the emerging pathogen Clostridioides (Clostridium) difficile might lead to colonization of the gastrointestinal tract of humans and mammals eventually resulting in antibiotic-associated diarrhea, which can be mild to possibly life-threatening. Recurrences after antibiotic treatment have been described in 15–30% of the cases and are either caused by the original (relapse) or by new strains (reinfection).In this study, we describe a patient with ongoing recurrent C. difficile infections over 13 months. During this time, ten C. difficile strains of six different ribotypes could be isolated that were further characterized by phenotypic and genomic analyses including motility and sporulation assays, growth fitness and antibiotic susceptibility as well as whole-genome sequencing. PCR ribotyping of the isolates confirmed that the recurrences were a mixture of relapses and reinfections. One recurrence was due to a mixed infection with three different strains of two different ribotypes.Furthermore, genomes were sequenced and multi-locus sequence typing (MLST) was carried out, which identified the strains as members of sequence types (STs) 10, 11, 14 and 76. Comparison of the genomes of isolates of the same ST originating from recurrent CDI (relapses) indicated little within-patient microevolution and some concurrent within-patient diversity of closely related strains.Isolates of ribotype 126 that are binary toxin positive differed from other ribotypes in various phenotypic aspects including motility, sporulation behavior and cell morphology. Ribotype 126 is genetically related to ribotype 078 that has been associated with increased virulence. Isolates of the ribotype 126 exhibited elongated cells and a chaining phenotype, which was confirmed by membrane staining and scanning electron microscopy. Furthermore, this strain exhibits a sinking behavior in liquid medium in stationary growth phase. Taken together, our observation has proven multiple CDI recurrences that were based on a mixture of relapses and reinfections.     

Distinct ribotypes and rates of antimicrobial drug resistance in Clostridium difficile from Shanghai and Stockholm

Seventy-five clinical isolates of Clostridium difficile from Shanghai and 80 from Stockholm were investigated. The prevalence of toxin A-negative, toxin B-positive isolates of C .  difficile among isolates from Shanghai (33.3%) was significantly higher than among isolates from Stockholm (0%). Both sets of isolates were fully susceptible to metronidazole and vancomycin. However, the MICs of fluoroquinolones, erythromycin–clindamycin, tetracycline, rifampin and fusidic acid were significantly higher for the Shanghai isolates than for the Stockholm isolates. Thirty-three PCR ribotypes were identified; a dominant clone, 017, accounted for 18.7% of Shanghai isolates, whereas clone 005 dominated among Stockholm isolates, accounting for 11.3%. Strains 027 and 078 were not detected. No outbreak occurred during the study period.     

Risk factors for recurrence of Clostridium difficile infection: effect of vancomycin-resistant enterococci colonization

Recurrent Clostridium difficile infection (CDI) is one of the most difficult problems in healthcare infection control. We evaluated the risk factors associated with recurrence in patients with CDI. A retrospective cohort study of 84 patients with CDI from December 2008 through October 2010 was performed at Pusan National University Yangsan Hospital. Recurrence occurred in 13.1% (11/84) of the cases and in-hospital mortality rate was 7.1% (6/84). Stool colonization with vancomycin-resistant enterococci (VRE) (P = 0.006), exposure to more than 3 antibiotics (P = 0.009), low hemoglobin levels (P = 0.025) and continued use of previous antibiotics (P = 0.05) were found to be more frequent in the recurrent group. Multivariate analysis indicated that, stool VRE colonization was independently associated with CDI recurrence (odds ratio, 14.519; 95% confidence interval, 1.157-182.229; P = 0.038). This result suggests that stool VRE colonization is a significant risk factor for CDI recurrence.     

Intravenous teicoplanin does not prevent Clostridium difficile associated diarrhea

A 59-year-old man with the diagnosis of endocarditis of the mitral valve due to Streptococcus mitis was treated with penicillin G, gentamicin, and later with clindamycin as inpatient for 3 weeks. Thereafter outpatient therapy with parenteral teicoplanin 3 × per week was initiated. After 17 days of teicoplanin treatment he developed severe diarrhea, and stool samples were positive for Clostridium difficile toxin. In addition to the ongoing parenteral therapy with teicoplanin, oral teicoplanin was administered. On the third day of this regimen the diarrhea and other disabling symptoms subsided, and test results for C. difficile toxin became negative. Oral teicoplanin was continued for 10 days and cleared C. difficile effectively after treatment as assessed by consecutive stool cultures (until 60 days thereafter). The parenteral administration of teicoplanin could not prevent the onset of C. difficile associated diarrhea in this patient, who previously had been treated with clindamycin. Thus, the administration of parenteral teicoplanin does not seem to be a treatment option for C. difficile associated diarrhea in patients in which oral therapy is not possible.     

Mathematical modeling of Clostridium difficile infection

Clostridium difficile diarrhea is a major cause of morbidity and mortality in hospitals. However, the number of cases in an outbreak is usually relatively small. This precludes many traditional statistical methods of modeling epidemics. Stochastic models are designed to deal with small numbers and are promising methods of understanding C. difficile epidemiology. This is illustrated by a reversible jump Markov chain Monte Carlo model based on the herd immunity hypothesis of C. difficile outbreaks.     

The pathogenicity of Clostridium difficile

It is now well established that the major virulence factors of C. difficile are the two toxins A and B. However, the organism possesses an array of other putative virulence factors that may be important for localisation within the colon, and in evasion of the immune system. It has been observed that certain types of C. difficile are more commonly found causing disease than others, and this seems to be independent of toxin production. Is this simply a reflection of their abundance in the hospital environment, or is it due to their virulence determinants? This review covers our current knowledge of the modes of action of toxins A and B at the cellular and molecular level. Many unanswered questions are posed that require answers before we can fully understand the pathogenic mechanisms of the organism and be in a position to manage better the spectrum of diseases it causes.     

Antecedent use of fluoroquinolones is associated with resistance to moxifloxacin in Clostridium difficile

Objective   Moxifloxacin is characterized by high activity against Gram-positive cocci and some Gram-positive and -negative anaerobes, including Clostridium difficile . This study investigates the role of prior quinolone use in relation to patterns of susceptibility of C. difficile to moxifloxacin.
Methods   Sixty-three clinical isolates of C. difficile were investigated for toxigenicity, susceptibility to moxifloxacin, and mutations in the DNA gyrase gene. The medical histories for 50 of these patients were available and used to identify previous fluoroquinolone use.
Results   Thirty-three (52.4%) strains showed resistance to moxifloxacin (MICs ≥ 16 mg/L). All moxifloxacin-resistant strains harbored a mutation at amino acid codon Ser-83 of gyrA . Forty-five isolates (71.4%) were toxigenic; all moxifloxacin-resistant strains were in this group. Resistance to moxifloxacin was associated with prior use of fluoroquinolones ( P -value 0.009, chi-square).
Conclusions   Although the use of moxifloxacin to treat C. difficile -associated diarrhea is not likely to be common, these data show a relationship between antecedent fluoroquinolone use and resistance to moxifloxacin in C. difficile isolates, and raise questions regarding selection pressure for resistance placed on colonizing bacteria exposed to fluoroquinolones. Mutations in gyrA are involved in moxifloxacin resistance.     

Detection of virulence genes of Clostridium difficile by multiplex PCR

Antikainen J, Pasanen T, Mero S, Tarkka E, Kirveskari J, Kotila S, Mentula S, Könönen E, Virolainen‐Julkunen A‐R, Vaara M, Tissari P. Detection of virulence genes of Clostridium difficile by multiplex PCR. APMIS 2009; 117: 607–13. 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.     

Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates

A 2-month prospective study of Clostridium difficile infections was conducted in 38 hospitals from 14 different European countries in order to obtain an overview of the phenotypic and genotypic features of clinical isolates of C. difficile during 2005. Of 411 isolates from diarrhoeagenic patients with suspected C. difficile-associated diarrhoea (CDAD), 354 were toxigenic, of which 86 (24.3%) were toxin-variant strains. Major toxinotypes included toxinotypes 0 (n = 268), V (n = 28), VIII (n = 22) and III (n = 25). MICs of metronidazole, vancomycin, erythromycin, clindamycin, moxifloxacin and tetracycline were determined using the Etest method. All the toxigenic strains were fully-susceptible to metronidazole and vancomycin. Resistance to erythromycin, clindamycin, tetracycline and moxifloxacin was found in 44.4%, 46.1%, 9.2% and 37.5% of the isolates, respectively. Sixty-six different PCR ribotypes were characterised, with the 027 epidemic strain accounting for 6.2% of isolates. This strain was positive for binary toxin genes, had an 18-bp deletion in the tcdC gene, and was resistant to both erythromycin and moxifloxacin. The mean incidence of CDAD was 2.45 cases/10 000 patient-days, but this figure varied widely among the participating hospitals. Patients infected with the 027 strain were more likely to have a severe disease (OR 3.29, 95% CI 1.19-9.16, p 0.008) and to have been specifically treated with metronidazole or vancomycin (OR 7.46, 95% CI 1.02-154, p 0.02). Ongoing epidemiological surveillance of cases of CDAD, with periodic characterisation of the strains involved, is required to detect clustering of cases in time and space and to monitor the emergence of specific highly virulent clones.     

Clostridium difficile infection: New insights into therapeutic options

Abstract

Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings and represents a major social and economic burden. The major virulence determinants are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), encoded within the pathogenicity locus. Traditional therapies, such as metronidazole and vancomycin, frequently lead to a vicious circle of recurrences due to their action against normal human microbiome. New disease management strategies together with the development of novel therapeutic and containment approaches are needed in order to better control outbreaks and treat patients. This article provides an overview of currently available CDI treatment options and discusses the most promising therapies under development.     

抗艰难梭菌A毒素单克隆抗体的制备及特性分析

目的 :制备抗艰难梭菌A毒素的单克隆抗体 (mAb)并鉴定其特性。方法 :用纯化的艰难梭菌A毒素免疫BALB/c小鼠 ,将免疫小鼠的脾细胞与骨髓瘤细胞Sp2 / 0融合 ,采用间接ELISA筛选杂交瘤细胞。用ELISA检测mAb腹水的效价、相对亲和力和进行表位分析 ;用Westernblot检测mAb的特异性。结果 :得到 6株杂交瘤细胞株 ,5C10株细胞分泌的mAb为IgG2a ,4B5和 8A1株细胞分泌的mAb为IgG1,其他 3株细胞mAb (2H7、3E9和 6G8)均分泌IgM。中和试验表明 ,所有的mAb均无中和活性。腹水mAb的效价均在 10 -4以上 ,其中mAb 2H7、6G8、5C10、4B5和 8A1具有共同的表位 ,而mAb 3E9识别的位点与其他 5株不同。mAb 8A1和 4B5的相对亲和力>10 5,其他 4株mAb的相对亲和力 >10 4。在非变性条件下 ,PAGE后Westernblot的结果显示 ,6株mAb均可与相对分子质量 (Mr)为 5 5× 10 4的A毒素产生反应 ;而在变性条件下 ,还原与非还原SDS PAGE后Westernblot均显示 ,6株mAb均可与Mr 为 5× 10 4～ 2 4× 10 4的A毒素产生反应。结论 :6株杂交瘤细胞株均能分泌抗艰难梭菌A毒素的特异性mAb ,为艰难梭菌A毒素的研究提供了有利的工具     

Rapid detection of Clostridium difficile toxin A in stool specimens

Objective: To evaluate a rapid (15-min) enzyme immunoassay in the format of an individual cassette (ImmunoCard toxin A, Meridian, BMD, Marne-la-Vallée, France) for the detection of Clostridium difficile toxin A in stool specimens.
Methods: We compared this new test with the cytotoxicity assay using MRC-5 cells, the ToxA test (TechLab, BioWhittaker, Fontenay-sous-bois, France) and toxigenic culture for the diagnosis of C. difficile -associated diseases (CDAD). A total of 236 stool specimens collected from 220 patients was simultaneously tested with the four methods. Discordant results were resolved by reviewing patients' clinical records.
Results: The prevalence of CDAD was 13.9%. Test sensitivities and specificities were 100% and 99% respectively for the cytotoxicity assay, 87.5% and 100% for ImmunoCard toxin A, 77.4% and 100% for the ToxA test and 100% and 98% for toxigenic culture.
Conclusions: The ImmunoCard Toxin A is a very rapid, individual and easy-to-perform test for the diagnosis of CDAD. It provides same-day results and may be useful for both guiding appropriate treatment and controlling nosocomial spread of C. difficile.     

Transmission of Clostridium difficile spores in isolation room environments and through hospital beds

The aim of this study was to determine the dissemination of Clostridium difficile (CD) spores in a hospital setting where the potassium monopersulfate‐based disinfectant Virkon^TM was used for cleaning. In the initial part of the study, we sampled 16 areas of frequent patient contact in 10 patient rooms where a patient with CD infection (CDI) had been accommodated. In the second part of the study, we obtained samples from 10 patient beds after discharge of CDI patients, both before and after the beds were cleaned. In the first part, CDspores were isolated in only 30% of the rooms. In the second part, which focused on transmission to hospital beds, C. difficile was found in four of 10 beds either before or after cleaning. In conclusion, in both parts of the study, we demonstrated a moderate spread of CD spores to the environment despite routine cleaning procedures involving Virkon^TM.     

Extra-intestinal infections caused by Clostridium difficile

The objective of this paper was to investigate the incidence of extra-intestinal infections caused by Clostridium difficile. During a 10-year period, the microbiology laboratory of our institution isolated 2034 isolates of C. difficile . Of the 2034 isolates, 21 (1.08%) were obtained from extra-intestinal sources. This represents an incidence of extra-intestinal isolation of four cases per 100 000 admissions. We were able to review the records of 17 patients for our study. The isolates in 12 patients were obtained from structures or fluids anatomically close to the colon and included the following infections: peritonitis in five cases (three primary and two secondary), intra-abdominal abscesses in three patients and abdominal wound infections in four cases. The infections in the other five patients were not in the anatomic vicinity of the colon. They included one case with a brain abscess, two episodes of bacteremia and two cases of foot infections (one chronic osteomyelitis). In all but one case, C. difficile isolation was obtained as part of a polymicrobial flora. The isolates were frequently non-toxigenic and the extra-intestinal infections occurred without concomitant diarrhea or prior anti-microbial therapy. Out of the 17 patients, eight died and nine survived. Death could not be directly attributed to C. difficile in any of the cases. The isolation of C. difficile outside the intestinal tract is very uncommon. Its clinical significance should be interpreted with caution.     

How to detect Clostridium difficile variant strains in a routine laboratory

Toxin A-negative, toxin B-positive strains (A–/B+) are the best studied examples of Clostridium difficile variant strains. In addition, there are some other groups of variant C. difficile strains that produce both toxins (A+/B+) or are non-cytotoxic (A–/B–) but differ from the reference strain VPI 10463 in their toxin genes. Here we describe two simple methods (amplification of the tcd A gene and amplification of the binary toxin gene cdt A) which can be used in rapid screening for variant C. difficile strains in collections or in routine laboratories.