Epidemiology and diagnostic accuracy of Clostridium perfringens toxins in the intestinal contents of camels, sheep, and cattle: a cross-sectional study in Dakahlia governorate, Egypt.

This study aimed to establish an accurate epidemiological surveillance tool for the detection of different C. perfringens types from 76 diseased and 34 healthy animals in Dakhalia Governorate, Egypt. A total of 110 intestinal content samples were randomly collected from camels, sheep, and cattle. C. perfringens was isolated and biochemically identified by the VITEK2 system. Toxinotyping and genotyping of C. perfringens isolates were specified by a multiscreen ELISA and real-time qPCR (rt-qPCR). The occurrence of C. perfringens was highest among camels (20% in healthy and 25% in diseased) and was lowest in cattle (23.1% and 14.7%). The cpa toxin was detected in all isolates by rt-qPCR and in 7 isolates by ELISA, ext toxin was detected in 7 isolates by rt-qPCR and in 6 isolates by ELISA, and cpb toxin was detected in 2 isolates by both rt-qPCR and ELISA. Four types of C. perfringens were identified by rt-qPCR, type A (65.2%), B (4.3%), C (4.3%), and D (26.1%), and three types by ELISA, type D (17.4%), A (8.7%) and C (4.3%). Our study indicated the prevalence of infection in Dakahlia by C. perfringens type A and D, particularly camels, and recommends adopting an appropriate vaccination strategy among the studied animals.

Clostridioides difficile: Characterization of the circulating toxinotypes in an Argentinean public hospital.

Clostridioides difficile is a spore-forming anaerobe microorganism associated to nosocomial diarrhea. Its virulence is mainly associated with TcdA and TcdB toxins, encoded by their respective tcdA and tcdB genes. These genes are part of the pathogenicity locus (PaLoc). Our aim was to characterize relevant C. difficile toxinotypes circulating in the hospital setting. The tcdA and tcdB genes were amplified and digested with different restriction enzymes: EcoRI for tcdA; HincII and AccI for tcdB. In addition, the presence of the cdtB (binary toxin) gene, TcdA and TcdB toxins by dot blot and the cytotoxic effect of culture supernatants on Vero cells, were evaluated. Altogether, these studies revealed three different circulating toxinotypes according to Rupnik's classification: 0, I and VIII, being the latter the most prevalent one. Even though more studies are certainly necessary (e.g. sequencing analysis), it is worth noting that the occurrence of toxinotype I could be related to the introduction of bacteria from different geographical origins. The multivariate analysis conducted on the laboratory values of individuals infected with the most prevalent toxinotype (VIII) showed that the isolates associated with fatal outcomes (GCD13, GCD14 and GCD22) are located in regions of the biplots related to altered laboratory values at admission. In other patients, although laboratory values at admission were not correlated, levels of urea, creatinine and white blood cells were positively correlated after the infection was diagnosed. Our study reveals the circulation of different toxinotypes of C. difficile strains in this public hospital. The variety of toxinotypes can arise from pre-existing microorganisms as well as through the introduction of bacteria from other geographical regions. The existence of microorganisms with different pathogenic potential is relevant for the control, follow-up, and treatment of the infections.

Isolation, toxinotyping and antimicrobial susceptibility testing of Clostridium perfringens isolated from Pakistan poultry.

BACKGROUND AND OBJECTIVES: Clostridium perfringens is a causative agent of enteric infections in animals including poultry by producing twenty different types of toxins. A single strain produces only a subset of these toxins, which form the basis of its classification into seven toxinotypes (A-G). C. perfringens toxinotype A is a widespread cause of necrotic enteritis (NE) in poultry. The current study was conducted to determine the prevalence of different toxins and antimicrobial susceptibility of C. perfringens isolated from Pakistan NE affected poultry. METHODS: A total of 134 intestinal samples of the diseased birds were collected postmortem and processed for isolation of C. perfringens using tryptose sulphite cycloserine (TSC) agar supplemented with d-cycloserine. Isolates were confirmed by Gram's staining, biochemical and molecular analyses. Toxinotyping was performed by multiplex PCR. Antimicrobial susceptibility profile of isolates was performed by Kirby Bauer disc diffusion method. RESULTS: A total of 34 strains of C. perfringens were isolated from 134 samples with prevalence rate of 25.37%. All the isolated strains were toxinotype A, as they were positive for alpha toxin (CPA) and negative for other tested toxins such as beta (CPB), epsilon (ETX), iota (ITX), enterotoxin (CPE), toxin perfringens large (TpeL) and necrotic B-like toxin (NetB). Interestingly, all the isolated strains of C. perfringens were multidrug resistant. The highest resistance was observed against Neomycin, Trimethoprim, Tetracycline and Lincomycin which are routinely used at Pakistan poultry production. CONCLUSION: C. perfringens toxinotype A is prevalent in Pakistan poultry. Incidence of C. perfringens with prevalence rate of 25.37% can pose serious threat to Pakistan's poultry industry given that all the isolated strains were multidrug resistant. Our findings highlight the need for new antibiotics and antibiotic alternatives to overcome multidrug resistance.

Insights on toxin genotyping, virulence, antibiogram profiling, biofilm formation and efficacy of disinfectants on biofilms of Clostridium perfringens isolated from poultry, animals and humans.

AIMS: This study aimed to determine the toxin genotypes, virulence determinants and antibiogram of Clostridium perfringens isolated from poultry, animals and humans. Biofilm formation and the efficacy of disinfectants on C. perfringens biofilms were studied. METHODS AND RESULTS: Thirty C. perfringens isolates (20 clinical and 10 from chicken carcasses) were genotyped by PCR and all isolates were genotype A (cpa+). The overall prevalence of cpe, cpb2, netB and tpeL virulence genes was 6·7, 56·7, 56·7 and 36·7% respectively. Twenty-one isolates (70%) were multidrug-resistant, 8 (26·7%) were extensive drug-resistant and one isolate (3·3%) was pan drug-resistant. The average multiple antibiotic resistance index was 0·7. Biofilms were produced by 63·3% of C. perfringens isolates and categorized as weak (36·7%), moderate (16·7%) and strong (10%). Sodium hypochlorite caused significant reduction in C. perfringens biofilms (P < 0·0001). CONCLUSIONS: All C. perfringens strains in this study were type A, resistant to multiple antibiotics and most of them were biofilm producers. Sodium hypochlorite showed higher efficacy in reducing C. perfringens biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reported the efficacy of disinfectants in reducing C. perfringens biofilms of economic and public health concern and recommends application on surfaces in farms, food processing plants and slaughterhouses.

Expansion of the Clostridium perfringens toxin-based typing scheme.

Clostridium perfringens causes many different histotoxic and enterotoxic diseases in humans and animals as a result of its ability to produce potent protein toxins, many of which are extracellular. The current scheme for the classification of isolates was finalized in the 1960s and is based on their ability to produce a combination of four typing toxins - α-toxin, ß-toxin, ε-toxin and ι-toxin - to divide C. perfringens strains into toxinotypes A to E. However, this scheme is now outdated since it does not take into account the discovery of other toxins that have been shown to be required for specific C. perfringens-mediated diseases. We present a long overdue revision of this toxinotyping scheme. The principles for the expansion of the typing system are described, as is a mechanism by which new toxinotypes can be proposed and subsequently approved. Based on these criteria two new toxinotypes have been established. C. perfringens type F consists of isolates that produce C. perfringens enterotoxin (CPE), but not ß-toxin, ε-toxin or ι-toxin. Type F strains will include strains responsible for C. perfringens-mediated human food poisoning and antibiotic associated diarrhea. C. perfringens type G comprises isolates that produce NetB toxin and thereby cause necrotic enteritis in chickens. There are at least two candidates for future C. perfringens toxinotypes, but further experimental work is required before these toxinotypes can formally be proposed and accepted.

Detection of toxigenic Clostridium perfringens and Clostridium botulinum from food sold in Lagos, Nigeria.

Food-borne diseases contribute to the huge burden of sickness and death globally and in the last decade, have become more frequently reported in Africa. In line with this, food safety is becoming a significant and growing public health problem in Nigeria. Diarrhoea is a common problem in Nigeria and has been reported but there has been little data on the possibility of clostridia as aetiological agents. Clostridium species are ubiquitous in the environment and in the gastrointestinal tract of man and animals and can serve as a marker for faecal contamination. We set out to determine the potential of these foods to transmit Clostridium species. A total of 220 food commodities from six local governments in Lagos State were sampled. Isolates obtained were identified based on cultural, morphological and biochemical characteristics. Toxinotyping was done using multiplex-PCR with primers specific for alpha, beta, epsilon and iota-toxin genes, enterotoxigenic cpe gene and neurotoxigenic BoNt gene. Fifty (22.7%) clostridial species were isolated of which 29 (58%) were identified as C. perfringens. Toxinotyping of the 29 strains showed that 28 (96.6%) were toxin producing C. perfringens type A while one (3.4%) was C. perfringens type D. Two (4%) C. botulinum species were isolated and identified by 16S rRNA sequencing, both harbouring BoNt/A gene. The contamination rates of food with Clostridium species show that food hygiene is a problem and Clostridium species may be a source of food borne disease in Lagos State, Nigeria.

Toxinotyping and antimicrobial susceptibility of enterotoxigenic Clostridium perfringens isolates from mutton, beef and chicken meat.

A total of 300 meat samples comprising mutton, beef, and chicken meat (n = 100) collected from either local butcher shops or large meat outlets situated at various areas of Lahore City located in Punjab province of Pakistan were tested for the isolation of Clostridium perfringens. Prevalence of the organism was highest in the chicken (6 %) followed by mutton (5 %) and beef (1 %). Contamination level was high (10/150) in the samples collected from local butcher shops in comparison to the samples collected from large meat outlets (2/150). All of the raw meat samples were negative for the presence of alpha, beta and epsilon toxins of C. perfringens as detected through ELISA. Out of a total number of 12 isolates only half were capable of producing enterotoxins when cultured in trypticase glucose yeast (TGY) broth. Toxinotyping of the isolates showed that 3 were of type A while one each of the remaining three belonged to type B, C, and D. Antibiotic susceptibility testing of the toxin producing isolates revealed that C. perfringens were susceptible to chloramphenicol, ciprofloxacin, metronidazole, and ceftriaxone. All of the other drugs were relatively less effective with a least activity of amoxicillin against the isolates.

Alpha toxin production potential and antibiotic resistance patterns of clostridium perfringens isolates from meat samples.

Objective: This research aimed to analyze the prevalence, molecular characteristics, toxinotyping, alpha toxin production potential, and antibiotic resistance pattern of Clostridium perfringens (C. perfringens) isolates in meat samples collected from various sources. Methods: Sixty meat samples were screened for alpha toxin using Enzyme-Linked Immunosorbent Assay (ELISA), revealing a positivity rate of 13.3%, predominantly in raw poultry meat. Subsequent culturing on Perfringens agar identified nine samples harboring characteristic C. perfringens colonies, primarily isolated from raw poultry meat. Molecular confirmation through 16S rRNA gene amplification and sequencing authenticated twelve isolates as C. perfringens, with nine strains exhibiting genetic resemblance to locally isolated strains. Toxinotyping assays targeting alpha toxin-specific genes confirmed all nine isolates as type A C. perfringens, with no detection of beta or epsilon toxin genes. Hemolytic assays demonstrated varying alpha toxin production potentials among isolates, with accession number OQ721004.1 displaying the highest production capacity. Moreover, antibiotic resistance profiling revealed multi-drug resistance patterns among the isolates. Results: The study identified distinct clusters within C. perfringens strains, indicating variations. Phylogenetic analysis delineated genetic relatedness among strains, elucidating potential evolutionary paths and divergences. Conclusion: The findings underscore the need for robust surveillance and control measures to mitigate the risk of C. perfringens contamination in meat products, particularly in raw poultry meat. Enhanced monitoring and prudent antimicrobial stewardship practices are warranted in both veterinary and clinical settings to address the observed antibiotic resistance profiles and prevent foodborne outbreaks.

Ribotyping of Clostridioides difficile in the Liberec Regional Hospital: a tertiary health care facility.

The ribotyping of Clostridioides difficile is one of the basic methods of molecular epidemiology for monitoring the spread of C. difficile infections. In the Czech Republic, this procedure is mainly available in university hospitals. The introduction of ribotyping in a tertiary health care facility such as Liberec Regional Hospital not only increases safety in the facility but also supports regional professional development. In our study, 556 stool samples collected between June 2017 and June 2018 were used for C. difficile infection screening, followed by cultivation, toxinotyping, and ribotyping of positive samples. The toxinotyping of 96 samples revealed that 44.8% of typed strains could produce toxins A and B encoded by tcdA and tcdB, respectively. The ribotyping of the same samples revealed two epidemic peaks, caused by the regionally most prevalent ribotype 176 (n = 30, 31.3). C. difficile infection incidence ranged between 5.5 and 4.2 cases per 10,000 patient-bed days. Molecular diagnostics and molecular epidemiology are the two most developing parts of clinical laboratories. The correct applications of molecular methods help ensure greater safety in hospitals.

Toxinotype A Clostridium perfringens causing septicaemia with intravascular haemolysis: two cases and review of the literature.

BACKGROUND: Septicaemia with intravascular haemolysis is a rare, but often fatal, presentation of Clostridium perfringens infection. C. perfringens is a Gram-positive, anaerobic bacterium that can produce multiple toxins. Toxinotyping is not performed regularly. METHODS: This article describes two human cases of C. perfringens infections. Toxinotyping was performed using polymerase chain reaction (PCR). Additionally, a structured review of the literature was performed which searched specifically for cases of C. perfringens infection with haemolysis. RESULTS: Both cases were identified as toxinotype A strains and both cases were fatal. Also, both cases showed marked haemolysis during their clinical course, which is assumed to have played a significant role in their outcome. In total, 83 references were identified describing human C. perfringens infection with haemolysis. Mortality rates have been stable over the last 10 years at 80%. Toxinotyping has been performed in a total of six cases. Of the four cases analysed by PCR, all were identified as toxinotype A. CONCLUSIONS: Haemolytic C. perfringens infections are rare but are fatal in most cases. Toxinotyping is performed rarely. The authors advocate increased use of toxinotyping to gain insight into pathophysiology and more effective interventions.

Infection Dynamics of Clostridium perfringens Fingerprinting in Buffalo and Cattle of Punjab Province, Pakistan.

Clostridium perfringens produces core virulence factors that are responsible for causing hemorrhagic abomasitis and enterotoxemia making food, animals, and humans susceptible to its infection. In this study, C. perfringens was isolated from necropsied intestinal content of buffalo and cattle belonging to four major bovine-producing regions in the Punjab Province of Pakistan for the purpose offind out the genetic variation. Out of total 160 bovine samples (n: 160), thirty-three (n: 33) isolates of C. perfringens were obtained from buffalo (Bubales bubalis) and cattle (Bos indicus) that were further subjected to biochemical tests; 16S rRNA based identification and toxinotyping was done using PCR (Polymerase Chain Reaction) and PFGE (Pulse Field Gel Electrophoresis) pulsotypesfor genetic diversity. Occurrence of C. perfringens was found to be maximum in zone-IV (Bhakkar and Dera Ghazi Khan) according to the heatmap. Correlation was found to be significant and positive among the toxinotypes (α-toxin, and ε-toxin). Response surface methodology (RSM) via central composite design (CCD) and Box-Behnken design (BBD) demonstrated substantial frequency of C. perfringens based toxinotypes in all sampling zones. PFGE distinguished all isolates into 26 different pulsotypes using SmaI subtyping. Co-clustering analysis based on PFGE further decoded a diversegenetic relationship among the collected isolates. This study could help us to advance toward disease array of C. perfringens and its probable transmission and control. This study demonstrates PFGE patterns from Pakistan, and typing of C. perfringens by PFGE helps illustrate and mitigate the incidence of running pulsotypes.

Genetic Relatedness, Antibiotic Resistance, and Effect of Silver Nanoparticle on Biofilm Formation by Clostridium perfringens Isolated from Chickens, Pigeons, Camels, and Human Consumers.

In this study, we determined the prevalence and toxin types of antibiotic-resistant Clostridium perfringens in chicken, pigeons, camels, and humans. We investigated the inhibitory effects of AgNPs on biofilm formation ability of the isolates and the genetic relatedness of the isolates from various sources determined using RAPD-PCR. Fifty isolates were identified using PCR, and all the isolates were of type A. The cpe and cpb2 genes were detected in 12% and 56% of the isolates, respectively. The effect of AgNPs on biofilm production of six representative isolates indicated that at the highest concentration of AgNPs (100 µg/mL), the inhibition percentages were 80.8-82.8%. The RAPD-PCR patterns of the 50 C. perfringens isolates from various sources revealed 33 profiles and four clusters, and the discriminatory power of RAPD-PCR was high. Multidrug-resistant C. perfringens isolates are predominant in the study area. The inhibition of biofilm formation by C. perfringens isolates was dose-dependent, and RAPD-PCR is a promising method for studying the genetic relatedness between the isolates from various sources. This is the first report of AgNPs' anti-biofilm activity against C. perfringens from chickens, pigeons, camels, and humans, to the best of our knowledge.

Prevalence, Genotypic and Phenotypic Characterization and Antibiotic Resistance Profile of Clostridium perfringens Type A and D Isolated from Feces of Sheep (Ovis aries) and Goats (Capra hircus) in Punjab, Pakistan.

Clostridium perfringens poses a serious threat to small ruminants by causing moderate to severe enterotoxaemia. Due to its ability to produce a wide arsenal of toxins, it is ranked among the most prevalent and important pathogens in livestock. This study focused on the molecular characterization of different Clostridium perfringens types along with their antimicrobial resistance profile. An overall higher prevalence of C. perfringens (46.1%) was detected based on mPCR among sheep and goats (healthy and diseased) in the Punjab province, Pakistan. The majority of the isolates were characterized as type A (82%), followed by type D (18%). Among the isolates from diseased sheep and goats, 27% were positive for cpa, 49% for cpa and cpb2, 9% for cpa and etx, 15% for cpa, cpb2 and etx. In the case of isolates from healthy sheep and goats, 59% were positive for cpa, 34% for cpb2 and cpa, 4% for cpa and etx, and 3% for cpa, cpb2 and etx. The prevalence of the beta2 toxin gene in the diseased sheep and goat population was 64% as compared to 37% in healthy animals. All 184 isolates (100%) were sensitive to rifampin and ceftiofur; the majority (57%) was sensitive to teicoplanin, chloramphenicol, amoxicillin, linezolid and enrofloxacin. A lower proportion of isolates (43%) were sensitive to ciprofloxacin and only 14% were susceptible to erythromycin. The findings of this study highlight the higher prevalence of C. perfringens in small ruminants and indicate that detailed pathogenesis studies are necessary to understand the explicit role of various toxins in causing enteric infections in sheep and goats including how they might be exploited to develop vaccines against these diseases.

The Relative Role of Toxins A and B in the Virulence of Clotridioides difficile.

Most pathogenic strains of C. difficile possess two large molecular weight single unit toxins with four similar functional domains. The toxins disrupt the actin cytoskeleton of intestinal epithelial cells leading to loss of tight junctions, which ultimately manifests as diarrhea in the host. While initial studies of purified toxins in animal models pointed to toxin A (TcdA) as the main virulence factor, animal studies using isogenic mutants demonstrated that toxin B (TcdB) alone was sufficient to cause disease. In addition, the natural occurrence of TcdA-/TcdB+ (TcdA-/B+)mutant strains was shown to be responsible for cases of C. difficile infection (CDI) with symptoms identical to CDI caused by fully toxigenic (A+/B+) strains. Identification of these cases was delayed during the period when clinical laboratories were using immunoassays that only detected TcdA (toxA EIA). Our hospital laboratory at the time performed culture as well as toxA EIA on patient stool samples. A total of 1.6% (23/1436) of all clinical isolates recovered over a 2.5-year period were TcdA-/B+ variants, the majority of which belonged to the restriction endonuclease analysis (REA) group CF and toxinotype VIII. Despite reports of serious disease due to TcdA-/B+ CF strains, these infections were typically mild, often not requiring specific treatment. While TcdB alone may be sufficient to cause disease, clinical evidence suggests that both toxins have a role in disease.

Pathogenicity locus determinants and toxinotyping of Clostridioides difficile isolates recovered from Iranian patients.

Little is known about the toxin profiles, toxinotypes and variations of toxin Clostridioides difficile C (tcdC) in Iranian C. difficile isolates. A total of 818 stool specimens were obtained from outpatients (n = 45) and hospitalized patients (n = 773) in Tehran, Iran, from 2011 to 2017. The 44 C. difficile isolates were subjected to PCR of toxin C. difficile A (tcdA), toxin C. difficile B (tcdB), tcdA 3'-end deletion, toxinotyping and sequencing of the tcdC gene. Thirty-eight isolates (86.36%) were identified as tcdA and tcdB positive, and the remaining six isolates (13.63%) were nontoxigenic. All tcdA- and tcdB-positive isolates yielded an amplicon of 2535 bp by PCR for the tcdA 3' end. Fourteen (36.84%), seventeen (44.73%) and seven (18.43%) isolates belonged to wild-type, toxin C. difficile C subclone3 (tcdC-sc3) and tcdC-A genotype of tcdC, respectively. Thirty-one isolates (81.57%) belonged to toxinotype 0, and seven isolates (18.42%) were classified as toxinotype V. This study provides evidence for the circulation of historical and hypervirulent isolates in the healthcare and community settings. Furthermore, it was also demonstrated that the tcdC-A genotype and toxinotype V are not uncommon among Iranian C. difficile isolates.

Clostridioides difficile: Characterization of the circulating toxinotypes in an Argentinean public hospital / Clostridioides difficile: caracterización de los toxinotipos circulantes en un hospital público de Argentina

Abstract Clostridioides difficile is a spore-forming anaerobe microorganism associated to nosocomial diarrhea. Its virulence is mainly associated with TcdA and TcdB toxins, encoded by their respective tcdA and tcdB genes. These genes are part of the pathogenicity locus (PaLoc). Our aim was to characterize relevant C. difficile toxinotypes circulating in the hospital setting. The tcdA and tcdB genes were amplified and digested with different restriction enzymes: EcoRI for tcdA; HincII and AccI for tcdB. In addition, the presence of the cdtB (binary toxin) gene, TcdA and TcdB toxins by dot blot and the cytotoxic effect of culture supernatants on Vero cells, were evaluated. Altogether, these studies revealed three different circulating toxinotypes according to Rupnik's classification: 0, I and VIII, being the latter the most prevalent one. Even though more studies are certainly necessary (e.g. sequencing analysis), it is worth noting that the occurrence of toxinotype I could be related to the introduction of bacteria from different geographical origins. The multivariate analysis conducted on the laboratory values of individuals infected with the most prevalent toxinotype (VIII) showed that the isolates associated with fatal outcomes (GCD13, GCD14 and GCD22) are located in regions of the biplots related to altered laboratory values at admission. In other patients, although laboratory values at admission were not correlated, levels of urea, creatinine and white blood cells were positively correlated after the infection was diagnosed. Our study reveals the circulation of different toxinotypes of C. difficile strains in this public hospital. The variety of toxinotypes can arise from pre-existing microorganisms as well as through the introduction of bacteria from other geographical regions. The existence of microorganisms with different pathogenic potential is relevant for the control, follow-up, and treatment of the infections.

Resumen Clostridioides difficile es un anaerobio esporulado que se asocia con episodios de diarreas hospitalarias. Su virulencia se encuentra vinculada, principalmente, a las toxinas TcdA y TcdB, codificadas por sus respectivos genes, tcdA y tcdB, que son parte de un locus de patogenicidad (PaLoc). Nuestro objetivo fue caracterizar los toxinotipos de C. difficile circulantes en un hospital público. Los genes tcdA y tcdB fueron amplificados y digeridos con diferentes enzimas de restricción: EcoRI para tcdA; HincII y AccI para tcdB. Además, se evaluó la presencia de cdtB (gen de la toxina binaria B) y de las toxinas A y B (por dot blot), así como el efecto citotóxico de sobrenadantes de cultivo sobre células Vero. En conjunto, estos estudios revelaron tres toxinotipos circulantes según la clasificación de Rupnik: 0, I y VIII; el más prevalente fue el último. Aunque son necesarios más estudios (ej., secuenciación), es interesante notar que la presencia del toxinotipo I podría estar relacionada con la introducción de bacterias de diferente origen geográfico. En los pacientes infectados con el toxinotipo VIII, el análisis multivariante de los resultados de laboratorio mostró que los aislamientos asociados a decesos (GCD13, GCD14 y GCD22) estaban situados en regiones de los biplots relacionados con valores de laboratorio alterados al momento de la internación. En los otros pacientes, aunque no se observó correlación entre los valores de laboratorio al momento de la internación y la evolución clínica, los niveles de urea, creatinina y recuento de glóbulos blancos estuvieron correlacionados positivamente entre sí una vez diagnosticada la infección. Nuestro estudio revela la circulación de diferentes toxinotipos de C. difficile en un mismo hospital público. La variedad de toxinotipos puede originarse a partir de microorganismos preexistentes en la región, así como también por la introducción de bacterias provenientes de otras regiones geográficas. La existencia de microorganismos con diferente potencial patogénico es relevante para el control, el seguimiento y el tratamiento de las infecciones.

Toxinotyping and Sequencing of Clostridium difficile Isolates from Patients in a Tertiary Care Hospital of Northern India.

BACKGROUND: Clostridium difficile is an important cause of infectious colitis among hospitalized patients across the globe. The pathogenic potential of C. difficile in producing significant morbidity and mortality is mainly due to production of toxins A and B. The outbreaks of C. difficile infection (CDI) are due to changes in the genetic sequences of the organism. There is hardly any molecular study reported on the prevalent types of C. difficile strains in India. Toxinotyping and sequencing of locally circulating C. difficile isolates from patients presenting to our tertiary care center of North India were done. MATERIALS AND METHODS: C. difficile strains (n = 174) isolated from 1,110 fecal samples from patients with suspected CDI were subjected to toxinotyping and partial sequencing of tcdA and tcdB genes. Comparison of nucleotide sequences with reference C. difficile 630 strain using BLAST was made and translated into corresponding amino acid sequences by ExPASy. RESULTS AND DISCUSSION: Of 174 C. difficile isolates, 121 were toxigenic, belonging to toxinotype 0 (n = 76) and VIII (n = 45). Partial sequencing of toxin genes using bioinformatics approaches revealed changes in toxin A sequences of five (50%) C. difficile isolates, but the translated nucleotide sequences showed substitution in only three of them. No variation was seen in the toxin B nucleotide sequences. Interstrain variations were found in the clinical C. difficile isolates in our region. CONCLUSION: PCR amplified toxigenic genes followed by sequencing can help to identify genetic changes and pathogenicity of varied collection of C. difficile isolates.

Genomic diversity of Clostridium difficile strains.

Approaches to exploring Clostridium difficile genomic diversity have ranged from molecular typing methods to use of comparative genome microarrays and whole genome sequence comparisons. The C. difficile population structure is clonal and distributed into six clades, which correlate well with MLST STs (multilocus sequence types) and PCR ribotypes. However, toxigenic strains and strains with increased virulence are distributed throughout several clades. Here we summarize studies on C. difficile genomic diversity, with emphasis on phylogenetic aspects, epidemiological aspect and variability of some virulence factors.

Comparison of polymerase chain reaction ribotyping, toxinotyping and nutritional aspects of toxin production of Clostridium difficile strains.

Clostridium difficile (C. difficile) is the leading cause of infectious diarrhea in hospitals worldwide. Enterotoxin A (TcdA) and cytotoxin B (TcdB), have been identified as the main virulence factors of C. difficile. In China, data on polymerase chain reaction (PCR) ribotypes and abilities of hospital-derived C. difficile isolates to produce TcdA and TcdB are sparse. In this study, we identified 40 C. difficile isolates from the Taizhou hospital and investigated their PCR ribotypes based on the 16S-23S rRNA gene intergenic spacer region. The ability of different ribotypes to produce TcdA and TcdB was determined by immunochromatography and cytotoxicity assays, respectively. The effects of the nutritional status on the production of these toxins were also investigated.

Clostridium difficile binary toxin CDT: mechanism, epidemiology, and potential clinical importance.

Binary toxin (CDT) is frequently observed in Clostridium difficile strains associated with increased severity of C. difficile infection (CDI). CDT belongs to the family of binary ADP-ribosylating toxins consisting of two separate toxin components: CDTa, the enzymatic ADP-ribosyltransferase which modifies actin, and CDTb which binds to host cells and translocates CDTa into the cytosol. CDTb is activated by serine proteases and binds to lipolysis stimulated lipoprotein receptor. ADP-ribosylation induces depolymerization of the actin cytoskeleton. Toxin-induced actin depolymerization also produces microtubule-based membrane protrusions which form a network on epithelial cells and increase bacterial adherence. Multiple clinical studies indicate an association between binary toxin genes in C. difficile and increased 30-d CDI mortality independent of PCR ribotype. Further studies including measures of binary toxin in stool, analyses of CDI mortality caused by CDT-producing strains, and examination of the relationship of CDT expression to TcdA and TcdB toxin variants and PCR ribotypes are needed.