A decade of Clostridioides difficile infection: a constant challenge to maintain the status quo.

INTRODUCTION: Clostridioides difficile infection (CDI) is a leading cause of healthcare-associated (HA) diarrhoea. We retrospectively investigated data from a comprehensive, multidisciplinary C. difficile surveillance programme focusing on hospitalized patients in a tertiary Irish hospital over 10 years. METHODS: Data from 2012 to 2021 were extracted from a centralized database, including patient demographics, admission, case and outbreak details, ribotypes (RTs), and (since 2016) antimicrobial exposures and CDI treatments. Counts of CDI by origin of infection were explored using ꭓ2 analyses, Poisson regression was used to investigate trends in rates of CDI and possible risk factors. Time to recurrent CDI was examined by a Cox proportional hazards regression. RESULTS: Over 10 years, 954 CDI patients had a 9% recurrent CDI rate. CDI testing requests occurred in only 22% of patients. Most CDIs were HA (82.2%) and affected females (odds ratio: 2.3, P<0.01). Fidaxomicin significantly reduced the hazard ratio of time to recurrent CDI. No trends in HA-CDI incidence were observed despite key time-point events and increasing hospital activity. In 2021, community-associated (CA)-CDI increased. RTs did not differ for HA versus CA for the most common RTs (014, 078, 005 and 015). Average length-of-stay differed significantly between HA (67.1 days) and CA (14.6 days) CDI. CONCLUSION: HA-CDI rates remained unchanged despite key events and increased hospital activity, whereas by 2021, CA-CDI was at its highest in a decade. The convergence of CA and HA RTs, and the proportion of CA-CDI, question the relevance of current case definitions when increasingly patients receive hospital care without an overnight hospital stay.

Comparison of Whole-Genome Sequence-Based Methods and PCR Ribotyping for Subtyping of Clostridioides difficile.

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks the discriminatory power to study transmission and outbreaks in detail. New molecular methods have the capacity to differentiate better and provide standardized and interlaboratory exchangeable data. Using a well-characterized collection of diverse strains (N = 630; 100 unique ribotypes [RTs]), we compared the discriminatory power of core genome multilocus sequence typing (cgMLST) (SeqSphere and EnteroBase), whole-genome MLST (wgMLST) (EnteroBase), and single-nucleotide polymorphism (SNP) analysis. A unique cgMLST profile (more than six allele differences) was observed in 82 of 100 RTs, indicating that cgMLST could distinguish most, but not all, RTs. Application of cgMLST in two outbreak settings with RT078 and RT181 (known to have low intra-RT allele differences) showed no distinction between outbreak and nonoutbreak strains in contrast to wgMLST and SNP analysis. We conclude that cgMLST has the potential to be an alternative to CE-PCR ribotyping. The method is reproducible, easy to standardize, and offers higher discrimination. However, adjusted cutoff thresholds and epidemiological data are necessary to recognize outbreaks of some specific RTs. We propose to use an allelic threshold of three alleles to identify outbreaks.

Predominance of Clostridium difficile ribotypes 012, 027 and 046 in a university hospital in Chile, 2012.

In a 1-year survey at a university hospital we found that 20·6% (81/392) of patients with antibiotic associated diarrohea where positive for C. difficile. The most common PCR ribotypes were 012 (14·8%), 027 (12·3%), 046 (12·3%) and 014/020 (9·9). The incidence rate was 2·6 cases of C. difficile infection for every 1000 outpatients.

The FERMI free-electron lasers.

FERMI is a seeded free-electron laser (FEL) facility located at the Elettra laboratory in Trieste, Italy, and is now in user operation with its first FEL line, FEL-1, covering the wavelength range between 100 and 20 nm. The second FEL line, FEL-2, a high-gain harmonic generation double-stage cascade covering the wavelength range 20-4 nm, has also completed commissioning and the first user call has been recently opened. An overview of the typical operating modes of the facility is presented.

Emergence and spread of predominantly community-onset Clostridium difficile PCR ribotype 244 infection in Australia, 2010 to 2012.

We describe an Australia-wide Clostridium difficile outbreak in 2011 and 2012 involving the previously uncommon ribotype 244. In Western Australia, 14 of 25 cases were community-associated, 11 were detected in patients younger than 65 years, 14 presented to emergency/outpatient departments, and 14 to non-tertiary/community hospitals. Using whole genome sequencing, we confirm ribotype 244 is from the same C. difficile clade as the epidemic ribotype 027. Like ribotype 027, it produces toxins A, B, and binary toxin, however it is fluoroquinolone-susceptible and thousands of single nucleotide variants distinct from ribotype 027. Fifteen outbreak isolates from across Australia were sequenced. Despite their geographic separation, all were genetically highly related without evidence of geographic clustering, consistent with a point source, for example affecting the national food chain. Comparison with reference laboratory strains revealed the outbreak clone shared a common ancestor with isolates from the United States and United Kingdom (UK). A strain obtained in the UK was phylogenetically related to our outbreak. Follow-up of that case revealed the patient had recently returned from Australia. Our data demonstrate new C. difficile strains are an on-going threat, with potential for rapid spread. Active surveillance is needed to identify and control emerging lineages.

Impact of non-Gaussian electron energy heating upon the performance of a seeded free-electron laser.

Laser-heater systems have been demonstrated to be an important component for the accelerators that drive high gain free electron laser (FEL) facilities. These heater systems suppress longitudinal microbunching instabilities by inducing a small and controllable slice energy spread to the electron beam. For transversely uniform heating, the energy spread augmentation is characterized by a non-Gaussian distribution. In this Letter, we demonstrate experimentally that in addition to suppression of the microbunching instability, the laser heater-induced energy distribution can be preserved to the FEL undulator entrance, significantly impacting the performance of high-gain harmonic generation (HGHG) FELs, especially at soft x-ray wavelengths. In particular, we show that the FEL intensity has several local maxima as a function of the induced heating caused by the non-Gaussian energy distribution together with a strong enhancement of the power at high harmonics relative to that expected for an electron beam with an equivalent Gaussian energy spread at an undulator entrance. These results suggest that a single stage HGHG FEL can produce scientifically interesting power levels at harmonic numbers m ≥ 25 and with current seed laser technology could reach output photon energies above 100 eV or greater.

Experimental demonstration of electron longitudinal-phase-space linearization by shaping the photoinjector laser pulse.

Control of the electron-beam longitudinal-phase-space distribution is of crucial importance in a number of accelerator applications, such as linac-driven free-electron lasers, colliders and energy recovery linacs. Some longitudinal-phase-space features produced by nonlinear electron beam self- fields, such as a quadratic energy chirp introduced by geometric longitudinal wakefields in radio-frequency (rf) accelerator structures, cannot be compensated by ordinary tuning of the linac rf phases nor corrected by a single high harmonic accelerating cavity. In this Letter we report an experimental demonstration of the removal of the quadratic energy chirp by properly shaping the electron beam current at the photoinjector. Specifically, a longitudinal ramp in the current distribution at the cathode linearizes the longitudinal wakefields in the downstream linac, resulting in a flat electron current and energy distribution. We present longitudinal-phase-space measurements in this novel configuration compared to those typically obtained without longitudinal current shaping at the FERMI linac.

Escherichia coli bacteraemia: 2 years of prospective regional surveillance (2010-12).

OBJECTIVES: To determine the prevalence of antibiotic resistance and the epidemiology of Escherichia coli bacteraemia isolates across the Yorkshire and Humber National Health Service region over 2 years. METHODS: Ten percent of all E. coli blood culture isolates were collected per month from 14 laboratories across the Yorkshire and Humber region. Individual laboratories submitted antibiotic susceptibility data and isolates were re-tested centrally using the VITEK2(®) system (bioMérieux, France). Isolates were also characterized using PCR to test for the presence of sequences encoding extended-spectrum ß-lactamases (ESBLs) and genotyped using amplified fragment length polymorphism (AFLP). Selected isolates were further characterized using multilocus sequence typing. RESULTS: Between July 2010 and June 2012, 770 isolates were examined: 63%, 40%, 14% and 7% of isolates were non-susceptible to ampicillin, trimethoprim, ciprofloxacin and gentamicin, respectively. Eight percent of isolates (n = 63) were ESBL positive; CTX-M group 1 enzymes were the most common (68%). There was a fluctuating trend in the prevalence of resistance to amoxicillin/clavulanic acid (MIC >8 mg/L): July-September 2010, 16%; July-September 2011, 38%; and April-June 2012, 22%. AFLP identified 106 types. The majority of isolates belonged to one of two AFLP types: AFLP 1 [sequence type (ST) 131; 17%] and AFLP 2 (ST73; 18%). ST131 and ST73 were both associated with hospital- and community-onset bacteraemia, and with urinary, hepatobiliary and gastrointestinal sources of infection. ESBL-positive isolates were predominantly ST131 (60%). CONCLUSIONS: Continued surveillance of antibiotic resistance among E. coli bacteraemia isolates is necessary to enhance these early baseline data. The variable prevalence of resistance to amoxicillin/clavulanic acid raises concerns, as both E. coli bacteraemia and empirical use of this antibiotic are common.

Comparison of multilocus variable-number tandem-repeat analysis and whole-genome sequencing for investigation of Clostridium difficile transmission.

No study to date has compared multilocus variable-number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS) in an investigation of the transmission of Clostridium difficile infection. Isolates from 61 adults with ongoing and/or recurrent C. difficile infections and 17 asymptomatic carriage episodes in children (201 samples), as well as from 61 suspected outbreaks affecting 2 to 41 patients in 31 hospitals in the United Kingdom (300 samples), underwent 7-locus MLVA and WGS in parallel. When the first and last samples from the same individual taken for a median (interquartile range [IQR]) of 63 days (43 to 105 days) apart were compared, the estimated rates of the evolution of single nucleotide variants (SNVs), summed tandem-repeat differences (STRDs), and locus variants (LVs) were 0.79 (95% confidence interval [CI], 0.00 to 1.75), 1.63 (95% CI, 0.00 to 3.59), and 1.21 (95% CI, 0.00 to 2.67)/called genome/year, respectively. Differences of >2 SNVs and >10 STRDs have been used to exclude direct case-to-case transmission. With the first serial sample per individual being used to assess discriminatory power, across all pairs of samples sharing a PCR ribotype, 192/283 (68%) differed by >10 STRDs and 217/283 (77%) by >2 SNVs. Among all pairs of cases from the same suspected outbreak, 1,190/1,488 (80%) pairs had concordant results using >2 SNVs and >10 STRDs to exclude transmission. For the discordant pairs, 229 (15%) had ≥2 SNVs but ≤10 STRDs, and 69 (5%) had ≤2 SNVs but ≥10 STRDs. Discordant pairs had higher numbers of LVs than concordant pairs, supporting the more diverse measure in each type of discordant pair. Conclusions on whether the potential outbreaks were confirmed were concordant in 58/61 (95%) investigations. Overall findings using MLVA and WGS were very similar despite the fact that they analyzed different parts of the bacterial genome. With improvements in WGS technology, it is likely that MLVA locus data will be available from WGS in the near future.

Two-colour pump-probe experiments with a twin-pulse-seed extreme ultraviolet free-electron laser.

Exploring the dynamics of matter driven to extreme non-equilibrium states by an intense ultrashort X-ray pulse is becoming reality, thanks to the advent of free-electron laser technology that allows development of different schemes for probing the response at variable time delay with a second pulse. Here we report the generation of two-colour extreme ultraviolet pulses of controlled wavelengths, intensity and timing by seeding of high-gain harmonic generation free-electron laser with multiple independent laser pulses. The potential of this new scheme is demonstrated by the time evolution of a titanium-grating diffraction pattern, tuning the two coherent pulses to the titanium M-resonance and varying their intensities. This reveals that an intense pulse induces abrupt pattern changes on a time scale shorter than hydrodynamic expansion and ablation. This result exemplifies the essential capabilities of the jitter-free multiple-colour free-electron laser pulse sequences to study evolving states of matter with element sensitivity.

Epidemiology of Clostridium difficile infections in a tertiary-care hospital in Korea.

To survey healthcare-associated Clostridium difficile infection (HA-CDI) in a 900-bed tertiary-care hospital, we prospectively investigated the epidemiology of CDI and distribution of PCR-ribotypes. From February 2009 through January 2010, all patients with HA-CDI were enrolled. Epidemiological information and prescription records for antibiotics were collected. The C. difficile isolates were characterized using reference strains and were tested for antibiotic susceptibility. During the survey, incidence of HA-CDI was 71.6 per 100 000 patient-days. In total, 140 C. difficile isolates were obtained from 166 patients with HA-CDI. The PCR-ribotyping yielded 38 distinct ribotypes. The three most frequently found ribotypes made up 56.4% of all isolates; they comprised 37 isolates (26.4%) of PCR-ribotype 018, 22 (15.7%) of toxin A-negative PCR-ribotype 017, and 20 (14.3%) of PCR-ribotype 001. Clostridium difficile PCR-ribotype 018 was present in all departments throughout the hospital during the 11 months, whereas ribotype 017 and ribotype 001 appeared mostly in the pulmonary department. Hypervirulent C. difficile PCR-ribotype 027 was detected in 1 month on two wards. The incidence of CDI in each department showed a seven-fold difference, which correlated significantly with the amount of prescribed clindamycin (R = 0.783, p 0.013) or moxifloxacin (R = 0.733, p 0.025) in the departments. The rates of resistance of the three commonest ribotypes to clindamycin and moxifloxacin were significantly higher than those of other strains (92.1% versus 38.2% and 89.5% versus 27.3%, respectively). CDI is an important nosocomially acquired infection and this study emphasizes the importance of implementing country-wide surveillance to detect and control CDI in Korea.

Morbidity and mortality associated with Clostridium difficile ribotype 078: a case-case study.

The morbidity and mortality associated with Clostridium difficile ribotype 078 were examined by comparison with other known outbreak strains. A healthcare interaction within eight weeks of a positive specimen significantly increased the likelihood of ribotype 078 compared with ribotype 027. Individuals with ribotype 078 also tended to come from community sources, have a hospital stay post specimen similar to ribotype 027 and a lower 30-day mortality, but these differences were not statistically significant. This study generates several hypotheses and a methodological platform to explore this unique profile.

Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England.

BACKGROUND: Marked increases in Clostridium difficile infection (CDI) incidence, driven by epidemic strain spread, is a global phenomenon. METHODS: The Clostridium difficile Ribotyping Network (CDRN) was established in 2007 as part of enhanced CDI surveillance in England, to facilitate the recognition and control of epidemic strains. We report on changes in CDI epidemiology in England in the first 3 years of CDRN. RESULTS: CDRN received 12,603 fecal specimens, comprising significantly (P < .05) increasing numbers and proportions of national CDI cases in 2007-2008 (n = 2109, 3.8%), 2008-2009 (n = 4774, 13.2%), and 2009-2010 (n = 5720, 22.3%). The C. difficile recovery rate was 90%, yielding 11,294 isolates for ribotyping. Rates of 9 of the 10 most common ribotypes changed significantly (P < .05) during 2007-2010. Clostridium difficile ribotype 027 predominated, but decreased markedly from 55% to 36% and 21% in 2007-2008, 2008-2009, and 2009-2010, respectively. The largest regional variations in prevalence occurred for ribotypes 027, 002, 015, and 078. Cephalosporin and fluoroquinolone use in CDI cases was reported significantly (P < .05) less frequently during 2007-2010. Mortality data were subject to potential reporting bias, but there was a significant decrease in CDI-associated deaths during 2007-2010, which may have been due to multiple factors, including reduced prevalence of ribotype 027. CONCLUSIONS: Access to C. difficile ribotyping was associated with significant changes in the prevalence of epidemic strains, especially ribotype 027. These changes coincided with markedly reduced CDI incidence and related mortality in England. CDI control programs should include prospective access to C. difficile typing and analysis of risk factors for CDI and outcomes.

Relatedness of human and animal Clostridium difficile PCR ribotype 078 isolates determined on the basis of multilocus variable-number tandem-repeat analysis and tetracycline resistance.

Totals of 102 and 56 Clostridium difficile type 078 strains of human and porcine origins, respectively, from four European countries were investigated by an optimized multilocus variable-number tandem-repeat analysis (MLVA) and for tetracycline susceptibility. Eighty-five percent of all isolates were genetically related, irrespective of human or porcine origin. Human strains were significantly more resistant to tetracycline than porcine strains. All tetracycline-resistant strains contained the Tn916-like transposon harboring the tet(M) gene. We conclude that strains from human and porcine origins are genetically related, irrespective of the country of origin. This may reflect a lack of diversity and/or common source.

Infection due to C. difficile ribotype 078: first report of cases in the Republic of Ireland.

Clostridium difficile is an important healthcare-associated pathogen. Hypervirulent strains such as those belonging to ribotype 027 have been widely reported in recent years. A second strain associated with hypervirulence is ribotype 078 and the prevalence of Clostridium difficile infection (CDI) due to this ribotype appears to be increasing. This report describes an outbreak, in which 15cases of CDI due to ribotype 078 were detected in an Irish hospital and from a nursing home in the hospital's catchment area. C. difficile ribotype 078 accounted for 15% of total isolates submitted for ribotyping. The average age of patients with CDI due to ribotype 078 was 76 years. Forty-six percent of patients experienced recurrence of symptoms within eight weeks of diagnosis and CDI was felt to have directly contributed to five of the eight deaths. Use of enhanced DNA fingerprinting identified clusters within the 15 cases and suggested hitherto unrecognised links between some patients with CDI. Such approaches offer the promise to delineate common sources and transmission routes for C. difficile.

The changing epidemiology of Clostridium difficile infections.

The epidemiology of Clostridium difficile infection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data. There have been clear changes in the clinical presentation, response to treatment, and outcome of CDI. These changes have been driven to a major degree by the emergence and epidemic spread of a novel strain, known as PCR ribotype 027 (sometimes referred to as BI/NAP1/027). We review the evidence for the changing epidemiology, clinical virulence and outcome of treatment of CDI, and the similarities and differences between data from various countries and continents. Community-acquired CDI has also emerged, although the evidence for this as a distinct new entity is less clear. There are new data on the etiology of and potential risk factors for CDI; controversial issues include specific antimicrobial agents, gastric acid suppressants, potential animal and food sources of C. difficile, and the effect of the use of alcohol-based hand hygiene agents.

A case-control study of community-associated Clostridium difficile infection.

OBJECTIVES: The aim of this study was to determine the incidence of and risk factors for community-associated Clostridium difficile infection (CDI). METHODS: Prospective surveillance of community-derived faecal samples for C. difficile cytotoxin, followed by a questionnaire-based case-control study in two distinct patient cohorts (one semi-rural and the other urban). RESULTS: The proportion of randomly selected faecal samples positive for C. difficile cytotoxin was 2.1% in both patient cohorts (median ages 73 and 45 years for the urban and semi-rural cohorts, respectively). Exposure to antibiotics in the previous 4 weeks, particularly multiple agents (P < 0.001), aminopenicillins (P < 0.05) and oral cephalosporins (P < 0.05), was significantly more frequent among cases than controls. Hospitalization in the preceding 6 months was significantly associated with CDI (45% versus 23%; P = 0.022). However, almost half the cases had not received antibiotic therapy in the month before C. difficile detection, and approximately one-third neither had exposure to antibiotics nor recent hospitalization. Contact with infants aged < or =2 years was significantly associated with CDI (14% versus 2%; P = 0.02). Prior exposure to gastrointestinal-acting drugs (proton pump inhibitor, H2 antagonist or non-steroidal anti-inflammatory) was not significantly more common in CDI cases. C. difficile PCR ribotype 001 caused 60% and 13% of urban and semi-rural community-associated CDI cases, respectively. CONCLUSIONS: Reliance on antibiotic history and age (> or =65 years) will contribute to missed diagnoses of community-associated CDI. Potential risk factors for community-associated CDI should be explored further to explain the large proportion of cases not linked to recent antibiotic therapy or hospitalization.

Use of highly discriminatory fingerprinting to analyze clusters of Clostridium difficile infection cases due to epidemic ribotype 027 strains.

We compared multilocus variable-number tandem-repeat analysis (MLVA) and macrorestriction endonuclease analysis using pulsed-field gel electrophoresis (PFGE) to determine their utility to identify clusters of Clostridium difficile infection (CDI) among 91 isolates of PCR ribotype 027 (NAP1, for North American pulsed-field type 1) from nine hospitals (and 10 general practitioners associated with one institution) in England. We also examined whether mortality in CDI cases was associated with specific MLVA subtypes. PFGE discriminated between ribotype 027 strains at >98% similarity, identifying five pulsovars (I to V) of 1 to 53 isolates. MLVA was markedly more discriminatory, identifying 23 types of 1 to 15 isolates (>71% similarity). PFGE pulsovars I and IV contained 14 and 8 MLVA types, respectively. Twenty-one of twenty-three (91%) of MLVA types were specific to individual PFGE pulsovars. Four CDI clusters were identified in institution A by conventional epidemiological analysis. MLVA typing identified two enlarged and two additional clusters. Thirty of forty-four (68%) patients in institution A with CDI caused by ribotype 027 strains were assigned to seven distinct clusters by a combination of MLVA typing and epidemiological records. Of 33 patients, comprising 14 different MLVA types, nine (27%) died by day 30 (early deaths). Eight of nine (89%) were associated with PFGE type IV C. difficile ribotype 027. Five of nine early deaths were associated with MLVA type 16, which was the dominant type in this cohort (10/33 cases); 4 other distinct MLVA types accounted for the other early deaths. MLVA was far superior to PFGE for analyzing clusters of CDI both within and between institutions. Further study is needed to examine whether subtypes of C. difficile ribotype 027 affect outcome.