Diversity of moxifloxacin resistance during a nosocomial outbreak of a predominantly ribotype ARU 027 Clostridium difficile diarrhea

To characterize the extent and diversity of moxifloxacin resistance among Clostridium difficile isolates recovered during a predominantly Anaerobe Reference Unit (ARU) ribotype 027-associated nosocomial outbreak of antibiotic associated diarrhea we measured the susceptibility of 34 field isolates and 6 laboratory strains of C. difficile to moxifloxacin. We ribotyped the isolates as well as assaying them by PCR for the metabolic gene, gdh, and the virulence genes, tcdA, tcdB, tcdC, cdtA and cdtB. All the laboratory isolates, including the historical ARU 027 isolate Cd196, were susceptible to moxifloxacin (≤2 μg/mL). 13 field isolates were susceptible to ≤2 μg/mL. Five were resistant to from 4 to 12 μg/mL (moderate resistance); 16 were resistant to ≥16 μg/mL (high resistance). We sequenced the quinolone resistance determining regions of gyrA (position 71-460) and gyrB (position 1059-1448) from two susceptible laboratory strains, all five isolates with moderate resistance and two highly resistant isolates. Two highly resistant isolates (Pitt 40, ribotype ARU 027 and Pitt 33, ribotype ARU 001) had the same C245T (Thr₈₂ΔIle) mutation. No other changes were seen. Amplification with primer pairs specific for the C245T mutant gyrA and for the wild type gene respectively confirmed all 16 highly resistant ARU 027 isolates, as well as the highly resistant isolates from other ribotypes, had the C245T mutation and that the mutation was absent from all other isolates. Among the five isolates with moderate resistance we found combinations of mutations within gyrA (T128A, Val₄₃ΔAsp and G349T, Ala₁₁₇ΔSer) and gyrB (G1276A, Arg₄₂₆ΔAsn). The G1396A (Glu₄₆₆ΔLys) mutation was not associated with increased resistance.     

C. difficile ribotype 027 or 176?

Clostridium difficile is a major nosocomial pathogen of present times. The analysis of 624 C. difficile strains from 11 hospitals in the Czech Republic in 2013 revealed that 40 % of isolates belonged to ribotype 176. These results suggest that the incidence of CDI (C. difficile infection) in the Czech Republic has increased probably in connection with C. difficile ribotype 176. The molecular systems Xpert C. difficile Epi assay (Cepheid Inc., Sunnyvale, CA) diagnoses toxigenic strains and supports C. difficile ribotype 027 determination based on three specific target places in the toxigenic C. difficile genome. Twenty-nine strains cultivated from stool specimens were evaluated by the Xpert systems as presumed C. difficile PCR ribotype 027 were confirmed as a C. difficile ribotype 176 based on ribotyping. A further 120 C. difficile strains of ribotype 176 were examined for the presence of genes tcdB, cdtB and deletion in position 117 in the tcdC gene. Our experience shows that due to the correspondence of the target places, C. difficile ribotype 176 may be interpreted as ribotype 027 by Xpert C. difficile Epi assay (Cepheid Inc., Sunnyvale, CA). Further molecular analysis as ribotyping based on capillary electrophoresis is needed to differentiate between C. difficile ribotypes 027 and 176 for appropriate epidemiological situation control on local and national levels.     

Bacteremia with a large clostridial toxin-negative,binary toxin-positive strain of Clostridium difficile

Bacteremia caused by Clostridium difficile is rare. In this report, we describe a case of C. difficile bacteremia caused by an unusual strain of C. difficile. The isolate contained neither toxin A nor B genes, however, binary toxin genes were present (tcdA⁻, tcdB⁻, cdtA⁺, cdtB⁺) and a 7.2-kb element unrelated to the PaLoc was found inserted within the PaLoc integration site. The clinical relevance of the isolate could not be determined.     

ICU-Onset Clostridium difficile Infection in a University Hospital in China: A Prospective Cohort Study

A prospective study was conducted to investigate the incidence, clinical profiles and outcome of ICU-onset CDI in a 50-bed medical ICU at a university hospital in China. Stools were collected from patients who developed ICU-onset diarrhea and was screened for tcdA (toxin A gene) and tcdB (toxin B gene) by PCR. CDI cases were compared with the ICU-onset non-CDI diarrhea cases for demographics, comorbidities, potential risk factors, major laboratory findings and outcomes. Stool samples from CDI cases were subjected to C. difficile culture and C. difficile isolates were screened for tcdA, tcdB and the binary toxin genes (cdtA and cdtB) using multiplex PCR. Strain typing of toxigenic C. difficile isolates was performed using multilocus sequence typing. There were 1,277 patients in the ICU during the study period and 124 (9.7%) developed ICU-onset diarrhea, of which 31 patients had CDI. The incidence of ICU-onset CDI was 25.2 cases per 10,000 ICU days. ICU-onset CDI cases had similar features with ICU-onset non-CDI diarrhea cases including the use of proton pump inhibitors and antibacterial agents. The crude mortality rate of ICU-onset CDI was 22.6%, but the attributable mortality rate of ICU-onset CDI was only 3.2% here. Toxigenic C. difficile isolates were recovered from 28 out of the 31 patients with CDI. cdtA and cdtB were found in two strains. Seventeen STs including 11 new STs were identified. All of the 11 new STs were single-locus variants of known STs and the 17 STs identified here could be clustered into 3 clades. The incidence of ICU-onset CDI here is similar to those in Europe and North America, suggesting that CDI is likely to be a common problem in China. Toxigenic C. difficile here belonged to a variety of STs, which may represent a significant clonal expansion rather than the true clonal diversity.     

Evaluation of a rapid molecular screening approach for the detection of toxigenic Clostridium difficile in general and subsequent identification of the tcdC Δ117 mutation in human stools

We have developed and validated a rapid molecular screening protocol for toxigenic Clostridium difficile, that also enables the identification of the hypervirulent epidemic 027/NAP1 strain. We describe a multiplex real-time PCR assay, which detects the presence of the tcdA and tcdB genes directly in stool samples. In case of positive PCR results, a separate multiplex real-time PCR typing assay was performed targeting the tcdC gene frame shift mutation at position 117. We prospectively compared the results of the screening PCR with those of a cytotoxicity assay (CTA), and a rapid immuno-enzyme assay for 161 stool samples with a specific request for diagnosis of C. difficile infection (CDI). A total of 16 stool samples were positive by CTA. The screening PCR assay confirmed all 16 samples, and gave a PCR positive signal in eight additional samples. The typing PCR assay detected the tcdC Δ117 mutation in 2/24 samples suggesting the presence of the epidemic strain in these samples. This was confirmed by PCR ribotyping and sequencing of the tcdC gene. Using CTA as the “gold standard”, the sensitivity, specificity, positive predictive value, and negative predictive value, for the screening PCR were 100%, 94.4%, 66.7%, and 100%, respectively. In conclusion, PCR may serve as a rapid negative screening assay for patients suspected of having CDI, although the low PPV hamper the use of PCR as a standalone test. However, PCR results may provide valuable information for patient management and minimising the spread of the epidemic 027/NAP1 strain.     

Clinical Clostridium difficile: clonality and pathogenicity locus diversity

Clostridium difficile infection (CDI) is an important cause of mortality and morbidity in healthcare settings. The major virulence determinants are large clostridial toxins, toxin A (tcdA) and toxin B (tcdB), encoded within the pathogenicity locus (PaLoc). Isolates vary in pathogenicity from hypervirulent PCR-ribotypes 027 and 078 with high mortality, to benign non-toxigenic strains carried asymptomatically. The relative pathogenicity of most toxigenic genotypes is still unclear, but may be influenced by PaLoc genetic variant. This is the largest study of C. difficile molecular epidemiology performed to date, in which a representative collection of recent isolates (n = 1290) from patients with CDI in Oxfordshire, UK, was genotyped by multilocus sequence typing. The population structure was described using NeighborNet and ClonalFrame. Sequence variation within toxin B (tcdB) and its negative regulator (tcdC), was mapped onto the population structure. The 69 Sequence Types (ST) showed evidence for homologous recombination with an effect on genetic diversification four times lower than mutation. Five previously recognised genetic groups or clades persisted, designated 1 to 5, each having a strikingly congruent association with tcdB and tcdC variants. Hypervirulent ST-11 (078) was the only member of clade 5, which was divergent from the other four clades within the MLST loci. However, it was closely related to the other clades within the tcdB and tcdC loci. ST-11 (078) may represent a divergent formerly non-toxigenic strain that acquired the PaLoc (at least) by genetic recombination. This study focused on human clinical isolates collected from a single geographic location, to achieve a uniquely high density of sampling. It sets a baseline of MLST data for future comparative studies investigating genotype virulence potential (using clinical severity data for these isolates), possible reservoirs of human CDI, and the evolutionary origins of hypervirulent strains.     

Tracing the Spread of Clostridium difficile Ribotype 027 in Germany Based on Bacterial Genome Sequences

We applied whole-genome sequencing to reconstruct the spatial and temporal dynamics underpinning the expansion of Clostridium difficile ribotype 027 in Germany. Based on re-sequencing of genomes from 57 clinical C. difficile isolates, which had been collected from hospitalized patients at 36 locations throughout Germany between 1990 and 2012, we demonstrate that C. difficile genomes have accumulated sequence variation sufficiently fast to document the pathogen''s spread at a regional scale. We detected both previously described lineages of fluoroquinolone-resistant C. difficile ribotype 027, FQR1 and FQR2. Using Bayesian phylogeographic analyses, we show that fluoroquinolone-resistant C. difficile 027 was imported into Germany at least four times, that it had been widely disseminated across multiple federal states even before the first outbreak was noted in 2007, and that it has continued to spread since.     

Molecular Epidemiology of Clostridium difficile at a Medical Center in Taiwan: Persistence of Genetically Clustering of A?B+ Isolates and Increase of A+B+ Isolates

Introduction

We investigated the changing trend of various toxigenic Clostridium difficile isolates at a 3 500-bed hospital in Taiwan. Genetic relatedness and antimicrobial susceptibility of toxigenic C. difficile isolates were also examined.

Methods

A total of 110 non-repeat toxigenic C. difficile isolates from different patients were collected between 2002 and 2007. Characterization of the 110 toxigenic isolates was performed using agar dilution method, multilocus variable-number tandem-repeat analysis (MLVA) genotyping, tcdC genotyping, and toxinotyping.

Results

Among the 110 toxigenic isolates studied, 70 isolates harbored tcdA and tcdB (A⁺B⁺) and 40 isolates harbored tcdB only (A⁻B⁺). The annual number of A⁺B⁺ isolates considerably increased over the 6-year study (P = 0.055). A total of 109 different MLVA genotypes were identified, in which A⁺B⁺ isolates and A⁻B⁺ isolates were differentiated into two genetic clusters with similarity of 17.6%. Twenty-four (60%) of the 40 A⁻B⁺ isolates formed a major cluster, MLVA-group 1, with a similarity of 85%. Seven (6.4%) resistant isolates were identified, including two metronidazole-resistant and five vancomycin-resistant isolates.

Conclusions

This study indicated a persistence of a MLVA group 1 A⁻B⁺ isolates and an increase of A⁺B⁺ isolates with diverse MLVA types. Moreover, C. difficile isolates with antimicrobial resistance to metronidazole or vancomycin were found to have emerged. Continuous surveillance is warranted to understand the recent situation and control the further spread of the toxigenic C. difficile isolates, especially among hospitalized patients.     

The Second Messenger Cyclic Di-GMP Regulates Clostridium difficile Toxin Production by Controlling Expression of sigD

The Gram-positive obligate anaerobe Clostridium difficile causes potentially fatal intestinal diseases. How this organism regulates virulence gene expression is poorly understood. In many bacterial species, the second messenger cyclic di-GMP (c-di-GMP) negatively regulates flagellar motility and, in some cases, virulence. c-di-GMP was previously shown to repress motility of C. difficile. Recent evidence indicates that flagellar gene expression is tightly linked with expression of the genes encoding the two C. difficile toxins TcdA and TcdB, which are key virulence factors for this pathogen. Here, the effect of c-di-GMP on expression of the toxin genes tcdA and tcdB was determined, and the mechanism connecting flagellar and toxin gene expressions was examined. In C. difficile, increasing c-di-GMP levels reduced the expression levels of tcdA and tcdB, as well as that of tcdR, which encodes an alternative sigma factor that activates tcdA and tcdB expression. We hypothesized that the C. difficile orthologue of the flagellar alternative sigma factor SigD (FliA; σ²⁸) mediates regulation of toxin gene expression in response to c-di-GMP. Indeed, ectopic expression of sigD in C. difficile resulted in increased expression levels of tcdR, tcdA, and tcdB. Furthermore, sigD expression enhanced toxin production and increased the cytopathic effect of C. difficile on cultured fibroblasts. Finally, evidence is provided that SigD directly activates tcdR expression and that SigD cannot activate tcdA or tcdB expression independent of TcdR. Taken together, these data suggest that SigD positively regulates toxin genes in C. difficile and that c-di-GMP can inhibit both motility and toxin production via SigD, making this signaling molecule a key virulence gene regulator in C. difficile.     

Molecular Characterization of Clostridium difficile Isolates from Human Subjects and the Environment

Clostridium difficile is a spore-forming, gram-positive, anaerobic bacillus that can cause C. difficile infection (CDI). However, only a few studies on the prevalence and antibiotic resistance of C. difficile in healthy individuals in China have been reported. We employed a spore enrichment culture to screen for C. difficile in the stool samples of 3699 healthy Chinese individuals who were divided into 4 groups: infants younger than 2 years of age and living at home with their parents; children aged 1 to 8 years of age and attending three different kindergarten schools; community-dwelling healthy adult aged 23–60 years old; and healthcare workers aged 28–80 years old. The C. difficile isolates were analyzed for the presence of toxin genes and typed by PCR ribotyping and multilocus sequence typing (MLST). The minimum inhibitory concentration of 8 antimicrobial agents was determined for all of the isolates using the agar dilution method. The intestinal carriage rate in the healthy children was 13.6% and ranged from 0% to 21% depending on age. The carriage rates in the 1654 community-dwelling healthy adults and 348 healthcare workers were 5.5% and 6.3%, respectively. Among the isolates, 226 were toxigenic (225 tcdA+/tcdB+ and 1 tcdA+/tcdB+ ctdA+/ctdB+). Twenty-four ribotypes were found, with the dominant type accounting for 29.7% of the isolates. The toxigenic isolates were typed into 27 MLST genotypes. All of the strains were susceptible to vancomycin, metronidazole, fidaxomicin, and rifaximin. High resistance to levofloxacin and ciprofloxacin at rates of 39.8% and 98.3%, respectively, were observed. ST37 isolates were more resistant to levofloxacin than the other STs. The PCR ribotypes and sequence types from the healthy populations were similar to those from the adult patients.     

Sensitivity to antibiotics of Clostridium difficile toxigenic nosocomial strains

Clostridium difficile is the etiological agent of diarrhoea and colitis, especially in elderly patients. The incidence of these diseases has increased during the last 10 years. Emergence of so-called hypervirulent strains is considered as one of the main factors responsible for the more severe disease and changed profile of sensitivity to antimicrobial agents. The aim of this work was to determine the sensitivity profile of toxigenic strains of C. difficile in the Czech Republic in 2011–2012 to selected antibiotics. The antibiotics clindamycin, metronidazole, vancomycin and amoxicillin with clavulanic acid were used for this purpose. Isolates cultured on Brazier's C. difficile selective agar were analysed for the presence of toxin genes using Xpert detection system. Xpert analysis revealed that 33 strains carried the genes for toxins tcdB, cdt and tcdCΔ117, thus showing characteristics typical for the hypervirulent ribotype 027/PFGE type NAP1/REA type B1. The remaining 29 strains carried only the gene for toxin B (tcdB) and not cdt and tcdCΔ117. Our results indicate the higher susceptibility of C. difficile hypertoxigenic strains to three out of four tested antibiotics (except vancomycin) than it is for the other toxigenic strains. We found that only 10.34 % of other toxigenic strains were resistant to clindamycin, and no resistance was found in all other cases. All the isolates were sensitive to amoxicillin/clavulanic acid in vitro. However, its use is not recommended for therapy of infections caused by C. difficile.     

Comparison of VIDAS CDAB and CDA immunoassay for the detection of Clostridium difficile in a tcdA− tcdB+ C. difficile prevalent area

Enzyme immunoassays for TcdA and/or TcdB are widely used for diagnosis of C. difficile infection. This study compared the performance of the new VIDAS C. difficile Toxin A & B assay (CDAB) with that of the existing VIDAS C. difficile Toxin A II assay (CDA) in a tcdA⁻tcdB⁺ prevalent area. A total of 555 fecal samples were cultured and tested using CDAB and CDA. C. difficile was isolated in 150 samples and the concordance rate was 81.8% (454/555) between CDAB and CDA. PCR assays for tcdA and/or tcdB were used as a confirmatory test on C. difficile strains recovered from culture positive cases (n = 150) and on fecal specimens in culture negative/CDAB positive or equivocal cases (n = 27). The number of tcdA⁺tcdB⁺, tcdA⁻tcdB⁺, and tcdA⁻tcdB⁻ strains on culture positive isolates (n = 150) were 75 (50.0%), 41 (27.3%), and 34 (22.7%), respectively. PCR assays for tcdB gene alone in stool specimens (n = 27) showed positivity in five cases. The sensitivity of VIDAS CDAB was higher than that of VIDAS CDA (65.3% vs. 29.8%), by more than 2-fold. The specificity of CDAB was almost the same as CDA (93.8% vs. 94.5%). Toxigenic culture of C. difficile isolates in culture positive/VIDAS CDAB negative cases (n = 62) additionally detected 22 VIDAS CDAB positive and 9 VIDAS CDAB equivocal cases. The VIDAS CDAB assay detects more tcdA⁺tcdB⁺ strains (60% vs. 45.3%) and tcdA⁻tcdB⁺ strains (70.7% vs. 0%) compared with VIDAS CDA.     

Spo0A Differentially Regulates Toxin Production in Evolutionarily Diverse Strains of Clostridium difficile

Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.     

An Economic Analysis of Strategies to Control Clostridium Difficile Transmission and Infection Using an Agent-Based Simulation Model

Background

A number of strategies exist to reduce Clostridium difficile (C. difficile) transmission. We conducted an economic evaluation of “bundling” these strategies together.

Methods

We constructed an agent-based computer simulation of nosocomial C. difficile transmission and infection in a hospital setting. This model included the following components: interactions between patients and health care workers; room contamination via C. difficile shedding; C. difficile hand carriage and removal via hand hygiene; patient acquisition of C. difficile via contact with contaminated rooms or health care workers; and patient antimicrobial use. Six interventions were introduced alone and "bundled" together: (a) aggressive C. difficile testing; (b) empiric isolation and treatment of symptomatic patients; (c) improved adherence to hand hygiene and (d) contact precautions; (e) improved use of soap and water for hand hygiene; and (f) improved environmental cleaning. Our analysis compared these interventions using values representing 3 different scenarios: (1) base-case (BASE) values that reflect typical hospital practice, (2) intervention (INT) values that represent implementation of hospital-wide efforts to reduce C. diff transmission, and (3) optimal (OPT) values representing the highest expected results from strong adherence to the interventions. Cost parameters for each intervention were obtained from published literature. We performed our analyses assuming low, normal, and high C. difficile importation prevalence and transmissibility of C. difficile.

Results

INT levels of the “bundled” intervention were cost-effective at a willingness-to-pay threshold of $100,000/quality-adjusted life-year in all importation prevalence and transmissibility scenarios. OPT levels of intervention were cost-effective for normal and high importation prevalence and transmissibility scenarios. When analyzed separately, hand hygiene compliance, environmental decontamination, and empiric isolation and treatment were the interventions that had the greatest impact on both cost and effectiveness.

Conclusions

A combination of available interventions to prevent CDI is likely to be cost-effective but the cost-effectiveness varies for different levels of intensity of the interventions depending on epidemiological conditions such as C. difficile importation prevalence and transmissibility.     

Diagnostic deficiencies of C. difficile infection among patients in a tertiary hospital in Saudi Arabia: A laboratory-based case series

Clostridioides difficile infection (CDI) has become a threatening public health problem in the developed world. In the kingdom of Saudi Arabia, prevalence of CDI is still unknown due to limited surveillance protocols and diagnostic resources. We used a two-step procedure to study and confirm C. difficile cases. We also studied toxin profiles of these isolates.Stool samples were collected from symptomatic patients and clinically suspected of CDI for almost 12 months. Isolates were confirmed by culture method followed by 16S rRNA sequencing. Multiplex PCR was performed for the identification of toxin A, toxin B and binary toxin genes and compared to Gene Expert results.Out of the 47 collected samples, 27 were successfully grown on culture media. 18 samples were confirmed as C. difficile by both culture and 16S rRNA sequencing. Interestingly, the rest of the isolates (9 species) belonged to different genera. Our results showed 95% of samples were positive for both toxin A and B (tcdA, tcdB) and all samples exhibited the toxin gene regulator tcdC. All samples were confirmed negative for the binary toxin gene ctdB and 11% of the isolates were positive for ctdA gene. Interestingly, one isolate harbored the binary toxin gene (cdtA⁺) and tested negative for both toxins A and B.We believe that combining the standard culture method with molecular techniques can make the detection of C. difficile more accurate.     

Development of TaqMan-Based Quantitative PCR for Sensitive and Selective Detection of Toxigenic Clostridium difficile in Human Stools

Background

Clostridium difficile is the main cause of nosocomial diarrhea, but is also found in asymptomatic subjects that are potentially involved in transmission of C. difficile infection. A sensitive and accurate detection method of C. difficile, especially toxigenic strains is indispensable for the epidemiological investigation.

Methods

TaqMan-based quantitative-PCR (qPCR) method for targeting 16S rRNA, tcdB, and tcdA genes of C. difficile was developed. The detection limit and accuracy of qPCR were evaluated by analyzing stool samples spiked with known amounts of C. difficile. A total of 235 stool specimens collected from 82 elderly nursing home residents were examined by qPCR, and the validity was evaluated by comparing the detection result with that by C. difficile selective culture (CDSC).

Results

The analysis of C. difficile-spiked stools confirmed that qPCR quantified whole C. difficile (TcdA⁺TcdB⁺, TcdA⁻TcdB⁺, and TcdA⁻TcdB⁻ types), TcdB-producing strains (TcdA⁺TcdB⁺ and TcdA⁻TcdB⁺ types), and TcdA-producing strains (TcdA⁺TcdB⁺ type), respectively, with a lower detection limit of 10³ cells/g of stool. Of the 235 specimens examined, 12 specimens (5.1%) were C. difficile-positive by qPCR: TcdA⁺TcdB⁺ strain in six specimens and TcdA⁻TcdB⁻ strain in the other six. CDSC detected C. difficile in 9 of the 12 specimens, and toxigenic types of the isolates from the 9 specimens were consistent with those identified by qPCR, supporting the validity of our qPCR method. Moreover, the qPCR examination revealed that the carriage rate of whole C. difficile and that of toxigenic strains in the 82 subjects over a 6-month period ranged from 2.4 to 6.8% and 1.2 to 3.8%, respectively. An average qPCR count of C. difficile detected was 10^4.5 cells/g of stool, suggesting that C. difficile constituted a very small fraction of intestinal microbiota.

Conclusion

Our qPCR method should be an effective tool for both clinical diagnosis and epidemiological investigation of C. difficile.     

Development of Photodynamic Antimicrobial Chemotherapy (PACT) for Clostridium difficile

Background

Clostridium difficile is the leading cause of antibiotic-associated diarrhoea and pseudo membranous colitis in the developed world. The aim of this study was to explore whether Photodynamic Antimicrobial Chemotherapy (PACT) could be used as a novel approach to treating C. difficile infections.

Methods

PACT utilises the ability of light-activated photosensitisers (PS) to produce reactive oxygen species (ROS) such as free radical species and singlet oxygen, which are lethal to cells. We screened thirteen PS against C. difficile planktonic cells, biofilm and germinating spores in vitro, and cytotoxicity of effective compounds was tested on the colorectal adenocarcinoma cell-line HT-29.

Results

Three PS were able to kill 99.9% of bacteria in both aerobic and anaerobic conditions, both in the planktonic state and in a biofilm, after exposure to red laser light (0.2 J/cm²) without harming model colon cells. The applicability of PACT to eradicate C. difficile germinative spores indirectly was also shown, by first inducing germination with the bile salt taurocholate, followed by PACT.

Conclusion

This innovative and simple approach offers the prospect of a new antimicrobial therapy using light to treat C. difficile infection of the colon.