Restriction Endonuclease Analysis Group Research Articles

Epidemiology of Clostridioides difficile infection at one hospital 10 years after an outbreak of the epidemic C. difficile strain BI/027: changing strain prevalence, antimicrobial susceptibilities, and patient antibiotic exposures.

In contrast to the epidemiology 10 years earlier at our hospital when the epidemic restriction endonuclease analysis (REA) group strain BI accounted for 72% of Clostridioides difficile isolates recovered from first-episode C. difficile infection (CDI) cases, BI represented 19% of first-episode CDI isolates in 2013-2015. Two additional REA group strains accounted for 31% of isolates (Y, 16%; DH, 12%). High-level resistance to fluoroquinolones and azithromycin was more common among BI isolates than among DH, Y, and non-BI/DH/Y isolates. Multivariable analysis revealed that BI cases were 2.47 times more likely to be associated with fluoroquinolone exposure compared to non-BI cases (95% confidence interval [CI]: 1.12-5.46). In addition, the odds of developing a CDI after third- or fourth-generation cephalosporin exposure was 2.83 times for DH cases than for non-DH cases (95% CI: 1.06-7.54). Fluoroquinolone use in the hospital decreased from 2005 to 2015 from a peak of 113 to a low of 56 antimicrobial days/1,000 patient days. In contrast, cephalosporin use increased from 42 to 81 antimicrobial days/1,000 patient days. These changes correlated with a decrease in geometric mean MIC for ciprofloxacin (61.03 to 42.65 mg/L, P = 0.02) and an increase in geometric mean MIC for ceftriaxone (40.87 to 86.14 mg/L, P < 0.01) among BI isolates. The BI strain remained resistant to fluoroquinolones, but an overall decrease in fluoroquinolone use and increase in cephalosporin use were associated with a decrease in the prevalence of BI, an increased diversity of C. difficile strain types, and the emergence of strains DH and Y.

Characteristics and outcomes of Clostridioides difficile infection after a change in the diagnostic testing algorithm.

Polymerase chain reaction (PCR) testing for the detection of C. difficile is a highly sensitive test. Some clinical laboratories have included a 2-step testing algorithm utilizing PCR plus toxin enzyme immunoassays (EIAs) to increase specificity. To determine the risk factors and outcomes of C. difficile PCR-positive/toxin-positive encounters compared to PCR-positive/toxin-negative encounters. Retrospective study. A Veterans' Affairs hospital. A retrospective case-control study of patient encounters with a positive C. difficile test by PCR and either a toxin EIA-positive assay (ie, cases) or toxin EIA-negative assay (ie, controls). Clinically relevant exposures and risk factors were determined to assess CDI recurrence at 30 days. Available encounter stool specimens were cultured for C. difficile and were subjected to restriction endonuclease analysis (REA) strain typing. Among 130 C. difficile PCR-positive patient encounters, 80 (61.5%) were toxin EIA negative and 50 (38.5%) were toxin EIA positive. Encounters that were toxin positive were more frequently treated (96.0%) compared to toxin-negative encounters (71.3%; P < .01). A multivariable logistic regression model revealed that toxin-negative encounters were less likely to suffer a recurrent CDI episode within 30 days (odds ratio [OR], 0.20, 95% confidence interval [CI], 0.05-0.83). Additionally, a higher C. difficile PCR cycle threshold predicted a lower risk of CDI recurrence at 30 days. (OR, 0.82; 95% CI, 0.68-0.98). During the study period, the REA group Y strain accounted for most toxin-negative encounters (32.5%; P = .05), whereas REA group BI strain accounted for most toxin-positive encounters (24.3%; P = .02). A testing strategy of PCR plus toxin EIA helped predict recurrent CDI.

Effect of the COVID-19 Pandemic on Rates and Epidemiology of Clostridioides difficile Infection in One VA Hospital.

The COVID-19 pandemic was associated with increases in some healthcare-associated infections. We investigated the impact of the pandemic on the rates and molecular epidemiology of Clostridioides difficile infection (CDI) within one VA hospital. We anticipated that the potential widespread use of antibiotics for pneumonia during the pandemic might increase CDI rates given that antibiotics are a major risk for CDI. Hospital data on patients with CDI and recurrent CDI (rCDI) were reviewed both prior to the COVID-19 pandemic (2015 to 2019) and during the pandemic (2020-2021). Restriction endonuclease analysis (REA) strain typing was performed on CD isolates recovered from stool samples collected from October 2019 to March 2022. CDI case numbers declined by 43.2% in 2020 to 2021 compared to the annual mean over the previous 5 years. The stool test positivity rate was also lower during the COVID-19 pandemic (14.3% vs. 17.2%; p = 0.013). Inpatient hospitalization rates declined, and rates of CDI among inpatients were reduced by 34.2% from 2020 to 2021. The mean monthly cases of rCDI also declined significantly after 2020 [3.38 (95% CI: 2.89-3.87) vs. 1.92 (95% CI: 1.27-2.56); p = <0.01]. Prior to the pandemic, REA group Y was the most prevalent CD strain among the major REA groups (27.3%). During the first wave of the pandemic, from 8 March 2020, to 30 June 2020, there was an increase in the relative incidence of REA group BI (26.7% vs. 9.1%); After adjusting for CDI risk factors, a multivariable logistic regression model revealed that the odds of developing an REA group BI CDI increased during the first pandemic wave (OR 6.41, 95% CI: 1.03-39.91) compared to the pre-pandemic period. In conclusion, the incidence of CDI and rCDI decreased significantly during the COVID-19 pandemic. In contrast, REA BI (Ribotype 027), a virulent, previously epidemic CD strain frequently associated with hospital transmission and outbreaks, reappeared as a prevalent strain during the first wave of the pandemic, but subsequently disappeared, and overall CDI rates declined.

Antimicrobial susceptibility of Clostridioides difficile to omadacycline and comparator antimicrobials.

Omadacycline is a novel aminomethylcycline tetracycline antimicrobial that was approved for the treatment of community-associated bacterial pneumonia (CABP) and acute bacterial skin and skin structure infections (ABSSSI) in 2018. Omadacycline has demonstrated a high degree of in vitro activity towards Clostridioides difficile and previous data have hypothesized that use of omadacycline for CABP or ABSSSI may decrease the risk of C. difficile infections. To compare the in vitro antimicrobial activity of omadacycline versus commonly used antimicrobials for the approved indications of use. We compared the antimicrobial activity of eight antimicrobials approved for CABP and ABSSSI against omadacycline by agar dilution on 200 clinically relevant contemporary C. difficile isolates representing local and national prevalent strain types. The in vitro omadacycline geometric mean MIC was 0.07 mg/L. Ceftriaxone resistance was noted in >50% of all isolates tested. The epidemic strain group, identified as restriction endonuclease analysis (REA) group BI, was commonly resistant to azithromycin (92%), moxifloxacin (86%) and clindamycin (78%). REA group DH strains had an elevated trimethoprim/sulfamethoxazole geometric mean MIC of 17.30 mg/L compared with the geometric mean MIC of 8.14 mg/L noted in all other isolates. In the REA group BK isolates that had a doxycycline MIC of ≥2 mg/L, the omadacycline MIC was <0.5 mg/L. Among 200 contemporary C. difficile isolates, there were no notable elevations in the in vitro omadacycline MIC, indicating a high level of activity towards C. difficile in comparison with commonly used antimicrobials for CABP and ABSSSI.

403. Strain Epidemiology of Clostridioides difficile across Three Geographically Distinct Medical Centers in Chicago

Abstract Background Clostridioides difficile infections (CDI) are caused by a large and diverse group of strains with differences in prevalence and associated morbidity. Over the past 20 years the C. difficile (CD) molecular epidemiology has changed as the prevalence of the epidemic strain recognized as restriction endonuclease analysis (REA) group BI or PCR-Ribotype group (RT) 027 has decreased. The objective of this study was to determine the current epidemiology of CD in the city of Chicago. Methods Baseline characteristics and symptoms were compared for 81 patients who tested positive for CD by PCR (tcdB) between 9/1/2021 and 10/7/2021 at 3 hospitals in the city of Chicago. Patients were classified as having healthcare-associated CDI (HA-CDI) if symptoms began &amp;gt;72 hours after hospital admission, community-associated CDI (CA-CDI) if symptoms began ≤72 hours prior to admission, and community-onset healthcare-associated CDI (COHA-CDI) if they had been hospitalized ≤4 weeks prior to CDI diagnosis. Available stools were cultured and recovered CD isolates underwent REA typing. Determination of CD colonization was made by review of symptoms including chronicity of symptoms, stool frequency, and response to treatment. Results Among all patients, 33% (27/81) were CA-CDI, 28% (23/81) COHA-CDI, 11% (9/81) HA-CDI, and 27% (22/81) were classified as colonized. Primary CDI accounted for 66% (39/59) of the infections. Among patients with a primary CDI, 46% (18/39) of patients were classified as CA-CDI whereas COHA-CDI and HA-CDI accounted for 54% (21/39) of infections. REA group Y was the most common group strain accounting for 29% (22/75) of isolates. (Figure 1) REA group Y accounted for 26% (7/27) of CA-CDI compared to 0 REA group BI [p=0.06], and REA group Y accounted for 35% (7/20) of all colonized patients. (Figure 2) Figure 1Figure 2 Conclusion There has been a marked change in the CD epidemiology within the city of Chicago since 2009 when REA group BI accounted for 61% of CDI (Black et al ICHE 2011; 32:897-902). REA group Y (typically identified as RT 014/020) is now the most common group strain in Chicago supplanting REA group BI (RT027). REA group Y appears to be associated primarily with CA-CDI and CD colonization. A detailed genomic analysis of REA group Y is required to determine potential reservoirs of REA group Y. Disclosures Mary K. Hayden, MD, Sanofi: Member, clinical adjudication panel Nicholas M. Moore, PhD, D(ABMM), Abbott Molecular: Grant/Research Support|Cepheid: Grant/Research Support Amanda Harrington, PhD, Beckman Coulter, Inc.: Clinical trial data collection funded by Beckman Coulter, Inc.|bioMeriuex/BioFire: Grant/Research Support Dale N. Gerding, MD, Destiny Pharma plc.: Advisor/Consultant Stuart Johnson, M.D., Ferring Pharmaceuticals: Membership on Ferring Publication Steering Committee|Ferring Pharmaceuticals: Employee|Summit Plc: Advisor/Consultant.

661. In vitro Activity of Omadacycline and Comparator Antibiotics against Clostridioides difficile

Abstract Background Omadacycline was approved for the treatment of community acquired bacterial pneumonia (CABP) and acute bacterial skin and skin structure infections (ABSSSI) in 2018. Previous studies demonstrate that omadacycline has in vitro activity against Clostridioides difficile. We determined the in vitro activity of omadacycline and of comparator antimicrobials for the approved indications, CABP and ABSSSI, towards a contemporary and clinically relevant collection of C. difficile isolates. Methods Antimicrobial agar dilution was performed on 200 clinical C. difficile isolates collected from 2014 – 2021. Isolates were selected based on the most prevalent restriction endonuclease analysis (REA) groups locally and nationally. Omadacycline was compared to 8 standard of care antimicrobials for CABP and ABSSSI: amoxicillin-clavulanate, azithromycin, ceftriaxone, clindamycin, doxycycline, linezolid, moxifloxacin, and trimethoprim-sulfamethoxazole. Results Omadacycline demonstrated in vitro activity against all C. difficile isolates tested. The geometric mean MIC of omadacycline was 0.07 μg/ml and the MIC50 and MIC90 were 0.0625 μg/ml and 0.125 μg/ml, respectively. Among the comparator antibiotics, 52% of isolates were resistant to ceftriaxone and 37% of isolates were resistant to clindamycin. The majority of REA group BI isolates were resistant to clindamycin (77.7%, 28/36) and the clindamycin geometric mean MICs were higher for group BI compared to all other REA group strains (32 µg/ml and 5.33 µg/ml, respectively, p &amp;lt; 0.005). REA group BI also had elevated azithromycin and moxifloxacin geometric MICs (335.2 and 17.3 µg/ml, respectively). REA group DH isolates had higher trimethoprim-sulfamethoxazole geometric MICs compared to all other REA group strains (17.28 µg/ml and 8.14 µg/ml, respectively p &amp;gt;0.001). Nearly half of REA group BK isolates (47%) had MICs ≥2 µg/ml which did not correspond to elevated omadacycline MICs for the same isolates. Conclusion Omadacycline demonstrated consistently low MICs against C. difficile when compared to approved antimicrobials for CABP and ABSSI which varied in activity among particular C. difficile strains. Omadacycline may reduce the risk of developing a C. difficile infection and requires further study. Disclosures Stuart Johnson, M.D., Ferring Pharmaceuticals: Membership on Ferring Publication Steering Committee|Ferring Pharmaceuticals: Employee|Summit Plc: Advisor/Consultant.

406. Vancomycin Activity against Clostridioides difficile Over Three Decades in Chicago

Abstract Background In 2017, the Infectious Diseases Society of America (IDSA) and Society for Healthcare Epidemiologist of America (SHEA) updated the C. difficile (CD) treatment guidelines recommending vancomycin as the preferred therapy for C. difficile infections (CDI). We assessed the in vitro vancomycin minimum inhibitory concentration (MIC) against CD across three decades to determine if the vancomycin MIC had increased since these guidelines were published. Methods We performed antimicrobial agar dilution susceptibility testing on 89 clinically relevant CD isolates collected within Chicagoland area. These isolates were selected from 3 separate timeframes: 2005 – 2007, 2013 – 2015, and 2021. Isolates were selected based on the prevalence of restriction endonuclease analysis (REA) strain types within each time cohort. Treatment response to vancomycin was reviewed for patients from the 2021 cohort if CD isolates had a vancomycin MIC of ≥ 16 μg/ml. Results The in vitro vancomycin geometric mean MIC against all 89 CD isolates was 2.53 μg/ml with a MIC50 of 2 μg/ml and MIC90 of 4 μg/ml. Comparing the 3 timeframes, the geometric mean vancomycin MICs from 2005-2007, 2013-2015, and 2021 were 2.35, 2.27, and 2.91, respectively (p=0.11). Comparison of the isolates collected from 2005 – 2015 to 2021, the in vitro vancomycin geometric mean MICs were 2.31 and 2.91, respectively (p = 0.037). REA group BI was the most common strain group to have an increased in vitro vancomycin MIC within the 2021 cohort as 4 of the 5 isolates tested had a MIC of 16 μg/ml. All 4 of the patients infected with these strains with an elevated vancomycin MICs were treated vancomycin. All 4 patients had a resolution of symptoms on vancomycin and 2 suffered from a recurrent infection within ≤4 weeks of completing vancomycin. Conclusion The vancomycin MIC against CD has trended upwards slightly over the past 20 years. The most notable change occurred between 2015 and 2021. We hypothesize that this increase is due to increased use of oral vancomycin for the treatment of CDI. However, these data indicate that most isolates still have a MIC of ≤4 μg/ml and an elevated MIC does not appear to impact clinical outcomes. Further study is required to determine if this upward trend in vancomycin MIC continues and if this could have any potential clinical implications. Disclosures Stuart Johnson, M.D., Ferring Pharmaceuticals: Membership on Ferring Publication Steering Committee|Ferring Pharmaceuticals: Employee|Summit Plc: Advisor/Consultant.

746. Clinical and Microbiological Characteristics of Clostridioides difficile Infection After a Change in Diagnostic Testing Algorithm

Abstract Background There is a lack of consensus on the most appropriate testing for C. difficile infection (CDI). The objective of this study was to determine the clinical and microbiological characteristics of CDI following a switch from stool PCR testing only to PCR reflexed to toxin testing. Figure 1. PCR Cycle Threshold Values Methods We reviewed the characteristics and outcomes of 50 consecutive patients who tested positive for CD by PCR (Xpert CD, Cepheid) between October 2019 and January 2020 at the Hines VA Hospital. Cases were defined by results of reflex toxin testing (Cdiff quick check complete, Alere/TechLab) after a positive PCR result. Baseline characteristics, symptoms, initial laboratory data, and treatments were compared as well as patient outcomes, including hospital readmission due to CDI at 30 days, and recurrent CDI (rCDI) at 30 and 90 days. The cycle threshold for the stool PCR result was recorded. Stools were cultured anaerobically for CD, and restriction endonuclease analysis (REA) strain typing was performed on the recovered CD isolates. Results Toxin testing was positive in 19/50 (38%) cases. Compared to stool toxin-negative cases, toxin-positive cases were older (95% vs. 71% were age ≥ 65, p = 0.06), more likely to have a history of CDI (37% vs. 23%, p = 0.34), and have ≥ 1 CDI episodes within 6 months (37% vs. 19%, p = 0.26). Treatment for CDI was more common in patients who had a positive toxin text. (95% vs 61%, p= 0.009). Among the 38 patients that received treatment, 33 received vancomycin (87%) and 8 patients (21%) had rCDI at 30 days. Of the 8 patients with rCDI, 2 were re-admitted to the hospital for CDI. The average PCR cycle threshold was lower in the toxin-positive stools compared to toxin-negative stools (24.46 and 29.96, p&amp;lt; 0.001; Fig. 1) The endemic REA group Y was the most common CD strain recovered (30%) and the previously epidemic and virulent REA group BI strain was recovered in 11% of the cases. Conclusion CDI cases diagnosed by positive stool PCR and positive toxin tests had more typical risk factors for CDI, a lower PCR cycle threshold and were more likely to have been treated for CDI. Outcomes were similar in this setting where infection with the virulent BI strain was uncommon. Disclosures Stuart Johnson, MD, Acurx Pharmaceuticals (Advisor or Review Panel member)Bio-K+ (Advisor or Review Panel member)Ferring Pharmaceutical (Advisor or Review Panel member)

Characterization of Clostridioides difficile isolates recovered from two Phase 3 surotomycin treatment trials by restriction endonuclease analysis, PCR ribotyping and antimicrobial susceptibilities.

To investigate the molecular epidemiology and antimicrobial susceptibility of Clostridioides difficile isolates from patients with C. difficile infection (CDI) from two Phase 3 clinical trials of surotomycin. In both trials [Protocol MK-4261-005 (NCT01597505) conducted across Europe, North America and Israel; and Protocol MK-4261-006 (NCT01598311) conducted across North America, Asia-Pacific and South America], patients with CDI were randomized (1:1) to receive oral surotomycin (250 mg twice daily) or oral vancomycin (125 mg four times per day) for 10 days. Stool samples were collected at baseline and C. difficile isolates were characterized by restriction endonuclease analysis (REA) and PCR ribotyping. Susceptibility testing was performed by agar dilution, according to CLSI recommendations. In total, 1147 patients were included in the microbiological modified ITT population. Of 992 recovered isolates, 922 (92.9%) were typed. There was a high association between REA groups and their corresponding predominant PCR ribotype (RT) for BI, DH, G and CF strains. REA group A showed more diverse PCR RTs. Overall, the most common strain was BI/RT027 (20.3%) followed by Y/RT014/020 (15.0%) and DH/RT106 (7.2%). The BI/RT027 strain was particularly prevalent in Europe (29.9%) and Canada (23.6%), with lower prevalence in the USA (16.8%) and Australia/New Zealand (3.4%). Resistance was most prevalent in the BI/RT027 strain, particularly to metronidazole, vancomycin and moxifloxacin. A wide variation in C. difficile strains, both within and across different geographical regions, was documented by both REA and ribotyping, which showed overall good correlation.

Sequential introduction of a multistep testing algorithm and nucleic acid amplification testing leading to an increase in Clostridioides difficile detection and a trend toward increased strain diversity.

Most clinical microbiology laboratories have replaced toxin immunoassay (EIA) alone with multistep testing (MST) protocols or nucleic acid amplification testing (NAAT) alone for the detection of C. difficile. Study the effect of changing testing strategies on C. difficile detection and strain diversity. Retrospective study. A Veterans' Affairs hospital. Initially, toxin EIA testing was replaced by an MST approach utilizing a glutamate dehydrogenase (GDH) and toxin EIA followed by tcdB NAAT for discordant results. After 18 months, MST was replaced by a NAAT-only strategy. Available patient stool specimens were cultured for C. difficile. Restriction endonuclease analysis (REA) strain typing and quantitative in vitro toxin testing were performed on recovered isolates. Before MST (toxin EIA), 79 of 708 specimens (11%) were positive, and after MST (MST-A), 121 of 517 specimens (23%) were positive (P < .0001). Prior to NAAT-only testing (MST-B), 80 of the 490 specimens (16%) were positive by MST, and after NAAT-only testing was implemented, 67 of the 368 specimens (18%) were positive (P = nonsignificant). After replacing toxin EIA testing, REA strain group diversity increased (8, 13, 13, and 10 REA groups in the toxin EIA, MST-A, MST-B, and NAAT-only periods, respectively) and in vitro toxin concentration decreased. The average log10 toxin concentration of the isolates were 2.08, 1.88, 1.20 and 1.55 ng/mL for the same periods, respectively. MST and NAAT had similar detection rates for C. difficile. Compared to toxin testing alone, they detected increased diversity of C. difficile strains, many of which were low toxin producing.

Unique Clindamycin-Resistant Clostridioides difficile Strain Related to Fluoroquinolone-Resistant Epidemic BI/RT027 Strain.

During a surveillance study of patients in a long-term care facility and the affiliated acute care hospital in the United States, we identified a Clostridioides difficile strain related to the epidemic PCR ribotype (RT) 027 strain associated with hospital outbreaks of severe disease. Fifteen patients were infected with this strain, characterized as restriction endonuclease analysis group DQ and RT591. Like RT027, DQ/RT591 contained genes for toxin B and binary toxin CDT and a tcdC gene of identical sequence. Whole-genome sequencing and multilocus sequence typing showed that DQ/RT591 is a member of the same multilocus sequence typing clade 2 as RT027 but in a separate cluster. DQ/RT591 produced a similar cytopathic effect as RT027 but showed delayed toxin production in vitro. DQ/RT591 was susceptible to moxifloxacin but highly resistant to clindamycin. Continued surveillance is warranted for this clindamycin-resistant strain that is related to the fluoroquinolone-resistant epidemic RT027 strain.

Correlation between restriction endonuclease analysis and PCR ribotyping for the identification of Clostridioides (Clostridium) difficile clinical strains

Restriction endonuclease analysis (REA) and PCR ribotyping are two typing systems that have been frequently utilized for molecular epidemiologic characterization of Clostridioides (Clostridium) difficile. To correlate typing data obtained from each method, we performed both REA and PCR ribotyping on a large and diverse set of historical and contemporary C. difficile infection clinical isolates. Eighty isolates were selected from each reference laboratory in the United States (Microbiology Reference Laboratory, Hines VA Medical Center) and United Kingdom (Clostridium difficile Network for England and Northern Ireland laboratory, University of Leeds). The 160 isolates were assigned to 82 unique ribotypes and 51 unique REA groups (116 unique REA types). In general, concordance between typing methods was good. Dendrogram analysis of PCR ribotype band patterns demonstrated close genetic relationships among strain types with discordant REA and ribotype assignments. While REA typing was more discriminatory, several REA types in this study were further discriminated by PCR ribotyping, indicating that discriminatory value of these typing methods may be strain dependent. These data will assist with molecular epidemiologic surveillance of strains identified by these two commonly used C. difficile typing systems.

Molecular epidemiology of Clostridioides (Clostridium) difficile strains recovered from clinical trials in the US, Canada and Europe from 2006-2009 to 2012-2015

The prevalence of C. difficile infection (CDI) and severe CDI are influenced by the prevalence of specific C. difficile strains, which are themselves influenced by antimicrobial susceptibility determinants as well as antimicrobial usage patterns. Restriction endonuclease analysis (REA) typing and antimicrobial susceptibility testing were used to characterize 1808 C. difficile isolates obtained from patients enrolled in four multicenter, multi-country, randomized CDI treatment trials conducted between 2006 and 2009 and between 2012 and 2015. By 2015, the epidemic REA group BI strain (RT027) had decreased in prevalence in North America (US: 43%–18%, Canada: 39%–24%, P < 0.001), but rates of moxifloxacin resistance remained high. In contrast, REA group Y (RT014/020) and DH (RT106) strains, both of which had low rates of moxifloxacin resistance, increased in prevalence (Y strain - US: 6%–17%, Canada: 11%–23%, P < 0.001; DH strain - US: 1%–11%, Canada: 0%–8%, P < 0.0001). In Europe, the BI strain (RT027) was highly prevalent in Eastern European countries in 2015, but was unchanged in other parts of Europe. As in North America, the Y strain (RT014/020) was prevalent in both time periods, but the DH strain was rarely identified. Continued international molecular surveillance of C. difficile will be important to track prevalence of known epidemic strains and detect emergence of new strains of potential epidemiologic significance.

Whole-genome analysis reveals the evolution and transmission of an MDR DH/NAP11/106 Clostridium difficile clone in a paediatric hospital.

Clostridium difficile strain DH/NAP11/106, a relatively antibiotic-susceptible strain, is now the most common cause of C. difficile infection (CDI) among adults in the USA. To identify mechanisms underlying the evolution and transmission of an MDR DH/NAP11/106 clone. WGS (Illumina MiSeq), restriction endonuclease analysis (REA) and antibiotic susceptibility testing were performed on 134 C. difficile isolates collected from paediatric patients with CDI over a 2 year period. Thirty-one of 134 (23%) isolates were REA group DH. Pairwise single-nucleotide variant (SNV) analyses identified a DH clone causing seven instances of CDI in two patients. During the 337 days between the first and second CDI, Patient 1 (P1) received 313 days of antibiotic therapy. Clindamycin and rifaximin resistance, and reduced vancomycin susceptibility (MIC 0.5-2 mg/L), were newly identified in the relapsed isolate. This MDR clone was transmitted to Patient 2 (P2) while P1 and P2 received care in adjacent private rooms. P1 and P2 each developed two additional CDI relapses. Comparative genomics analyses demonstrated SNVs in multiple antibiotic resistance genes, including rpoB (rifaximin resistance), gyrB and a gene encoding PBP; gyrB and PBP mutations did not consistently confer a resistance phenotype. The clone also acquired a 46 000 bp genomic element, likely a conjugative plasmid, which contained ermB (clindamycin resistance). The element shared 99% identity with the genomic sequence of Faecalibacterium prausnitzii, an enteric commensal. These data highlight the emergence of MDR in C. difficile strain DH/NAP11/106 through multiple independent mechanisms probably as a consequence of profound antibiotic pressure.

Differences in the Molecular Epidemiology and Antibiotic Susceptibility of Clostridium difficile Isolates in Pediatric and Adult Patients.

The rising incidence of Clostridium difficile infections (CDIs) in adults is partly related to the global spread of fluoroquinolone-resistant strains, namely, BI/NAP1/027. Although CDIs are also increasingly diagnosed in children, BI/NAP1/027 is relatively uncommon in children. Little is known about the antibiotic susceptibility of pediatric CDI isolates. C. difficile was cultured from tcdB-positive stools collected from children diagnosed with CDI between December 2012 and December 2013 at an academic children's hospital. CDI isolates were grouped by restriction endonuclease analysis (REA). MICs were measured by agar dilution method for 7 antibiotics. Susceptibility breakpoints were based on guidelines from CLSI and/or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). MICs and REA groupings of C. difficile isolates from 74 adult patients (29 isolates underwent REA) from a temporally and geographically similar adult cohort were compared to those of pediatric isolates. Among 122 pediatric and 74 adult isolates, respectively, the rates of resistance were as follows: metronidazole, 0% and 0%; vancomycin, 0% and 8% (P = 0.003); rifaximin, 1.6% and 6.7% (P = 0.11); clindamycin, 18.9% and 25.3% (P = 0.29); and moxifloxacin, 2.5% and 36% (P = <0.0001). Only 1 of 122 (0.8%) BI/NAP1/027 isolates was identified among the children, compared to 9 of 29 (31%) isolates identified among the adults (P = <0.0001). The 3 moxifloxacin-resistant pediatric isolates were of REA groups BI and CF and a nonspecific group. The 2 rifaximin-resistant pediatric isolates were of REA groups DH and Y. The 21 clindamycin-resistant pediatric isolates were distributed among 9 REA groups (groups A, CF, DH, G, L, M, and Y and 2 unique nonspecific REA groups). These data suggest that a diverse array of relatively antibiotic-susceptible C. difficile strains predominate in a cohort of children with CDI compared to adults.

Restriction Endonuclease Analysis Typing of Clostridium difficile Isolates.

Restriction endonuclease analysis (REA) typing using HindIII enzyme is a highly discriminatory, reproducible, and consistent method of genetic typing of Clostridium difficile (CD) isolates. REA typing analyzes CD whole cellular DNA on two levels of discrimination: REA Group designation and REA Type designation, which distinguishes specific subtypes within the REA Group. This methodology has enabled the tracking of epidemiologically significant CD strains over time and in some cases has allowed documentation of the evolution of previously rare REA Group strains that have subsequently become epidemic. The chapter details the methods used to isolate and purify CD colonies from stool samples, to obtain intact, full-length whole cellular DNA from CD isolates by use of guanidine-EDTA solution, and to analyze the HindIII-digested DNA after electrophoretic separation on agarose gels.

U.S.-Based National Sentinel Surveillance Study for the Epidemiology of Clostridium difficile-Associated Diarrheal Isolates and Their Susceptibility to Fidaxomicin.

In 2011 a surveillance study for the susceptibility to fidaxomicin and epidemiology of Clostridium difficile isolates in the United States was undertaken in seven geographically dispersed medical centers. This report encompasses baseline surveillance in 2011 and 2012 on 925 isolates. A convenience sample of C. difficile isolates or toxin positive stools from patients were referred to a central laboratory. Antimicrobial susceptibility was determined by agar dilution (CLSI M11-A8). Clinical and Laboratory Standards Institute (CLSI), Food and Drug Administration, or European Union of Clinical Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied where applicable. Toxin gene profiles were characterized by multiplex PCR on each isolate. A random sample of 322 strains, stratified by institution, underwent restriction endonuclease analysis (REA). The fidaxomicin MIC90 was 0.5 μg/ml for all isolates regardless of REA type or toxin gene profile, and all isolates were inhibited at ≤1.0 μg/ml. By REA typing, BI strains represented 25.5% of the isolates. The toxin gene profile of tcdA, tcdB, and cdtA/B positive with a tcdC 18-bp deletion correlated with BI REA group. Moxifloxacin and clindamycin resistance was increased among either BI or binary toxin-positive isolates. Metronidazole and vancomycin showed reduced susceptibility (EUCAST criteria) in these isolates. Geographic variations in susceptibility, REA group and binary toxin gene presence were observed. Fidaxomicin activity against C. difficile isolated in a national surveillance study did not change more than 1 year after licensure. This analysis provides baseline results for future comparisons.

Molecular epidemiology of Clostridium difficile infections in children: a retrospective cohort study.

The molecular epidemiology of pediatric Clostridium difficile infection (CDI) is poorly understood. We aimed to identify the restriction endonuclease analysis (REA) groups causing CDI and to determine risk factors and outcomes associated with CDI caused by epidemic strains in children. Retrospective cohort study. Inpatients and outpatients >1 year old receiving care between December 2012 and December 2013. An academic children's hospital in Chicago, Illinois. C. difficile PCR-positive stools were cultured, and C. difficile isolates were typed by REA. REA of isolates from patients with multiple CDIs was performed to differentiate relapse (infection with same strain) from reinfection (different strains) irrespective of time between CDIs. A total of 189 CDIs occurred among 145 patients. REA groups were widely distributed. The BI/NAP1/027 strain caused CDI in only 1 patient. DH/NAP11/106, the predominant epidemic strain identified, was associated with the use of third- or fourth-generation cephalosporins (risk ratio [RR], 3.2; 95% confidence interval [CI], 1.1-9.9; P=.04). CDI relapse commonly occurred up to 20 weeks later. Compared with CDI caused by non-DH/NAP11/106 strains, CDI caused by DH/NAP11/106 was more likely to result in multiple CDI relapses (40% vs 8%; P=.05) among children with multiple CDIs. REA identified the exceedingly low prevalence of BI/NAP1/027 and the high prevalence of DH/NAP11/106, a common epidemic strain in the United Kingdom that is less often reported in the United States. CDI relapse commonly occurred up to 20 weeks from the previous CDI. Defining recurrent CDI as that occurring only within 8 weeks of the original infection may lead to misclassification of some recurrent CDIs as new CDIs in children.

Surotomycin demonstrates low in vitro frequency of resistance and rapid bactericidal activity in Clostridium difficile, Enterococcus faecalis, and Enterococcus faecium.

Surotomycin (CB-183,315) is an orally administered, minimally absorbed, selective bactericidal cyclic lipopeptide in phase 3 development for the treatment of Clostridium difficile-associated diarrhea. The aim of this study was to evaluate the emergence of resistance in C. difficile (ATCC 700057 and three recent clinical isolates from the restriction endonuclease analysis groups BI, BK, and K), vancomycin-susceptible (VS) Enterococcus faecalis (ATCC 49452), vancomycin-resistant (VR) E. faecalis (ATCC 700802), VS Enterococcus faecium (ATCC 6569), and VR E. faecium (ATCC 51559) under anaerobic conditions. The rate of spontaneous resistance was below the limit of detection (<10(-8) to <10(-9)) for surotomycin at 16 and 32× the MIC for all isolates tested. Under selective pressure by serial passage, C. difficile grew in a maximum of 4 μg/ml surotomycin (final MICs of 2 to 8 μg/ml [4- to 16-fold higher than those of the naive control]) at day 15, with the exception of the C. difficile BK strain, which grew in 16 to 32 μg/ml (final MICs of 8 to 32 μg/ml [16- to 64-fold higher than those of the naive control]). Enterococci remained relatively unchanged over 15 days, growing in a maximum of 8 μg/ml surotomycin (final MICs of 2 to 16 μg/ml [8- to 64-fold higher than those of the naive control]). Of the isolates tested, no cross-resistance to vancomycin, rifampin, ampicillin, metronidazole, or moxifloxacin was observed. Surotomycin at 20× MIC demonstrated equally rapid bactericidal activity (≥ 3-log-unit reduction in CFU/ml in ≤ 8 h) against naive and reduced-susceptibility isolates of C. difficile, VS Enterococcus (VSE), and VR Enterococcus (VRE), except for C. difficile BK (2.6-log-unit reductions for both). These results suggest that emergence of resistance to surotomycin against C. difficile, E. faecalis, and E. faecium is likely to be rare.

Nontoxigenic Clostridium difficile Protects Hamsters against Challenge with Historic and Epidemic Strains of Toxigenic BI/NAP1/027 C. difficile

Nontoxigenic Clostridium difficile (NTCD) has been shown to prevent fatal C. difficile infection in the hamster model when hamsters are challenged with standard toxigenic C. difficile strains. The purpose of this study was to determine if NTCD can prevent C. difficile infection in the hamster model when hamsters are challenged with restriction endonuclease analysis group BI C. difficile strains. Groups of 10 hamsters were given oral clindamycin, followed on day 2 by 10(6) CFU of spores of NTCD strain M3 or T7, and were challenged on day 5 with 100 CFU of spores of BI1 or BI6. To conserve animals, results for control hamsters challenged with BI1 or BI6 from the present study and controls from previous identical experiments were combined for statistical comparisons. NTCD strains M3 and T7 achieved 100% colonization and were 100% protective against challenge with BI1 (P ≤ 0.001). M3 colonized 9/10 hamsters and protected against BI6 challenge in the colonized hamsters (P = 0.0003). T7 colonized 10/10 hamsters, but following BI6 challenge, cocolonization occurred in 5 hamsters, 4 of which died, for protection of 6/10 animals (P = 0.02). NTCD colonization provides protection against challenge with toxigenic BI group strains. M3 is more effective than T7 in preventing C. difficile infection caused by the BI6 epidemic strain. Prevention of C. difficile infection caused by the epidemic BI6 strain may be more challenging than that of infections caused by historic BI1 and non-BI C. difficile strains.