Fecal microbiota transplantation: current challenges and future landscapes.

SUMMARYGiven the importance of gut microbial homeostasis in maintaining health, there has been considerable interest in developing innovative therapeutic strategies for restoring gut microbiota. One such approach, fecal microbiota transplantation (FMT), is the main "whole gut microbiome replacement" strategy and has been integrated into clinical practice guidelines for treating recurrent Clostridioides difficile infection (rCDI). Furthermore, the potential application of FMT in other indications such as inflammatory bowel disease (IBD), metabolic syndrome, and solid tumor malignancies is an area of intense interest and active research. However, the complex and variable nature of FMT makes it challenging to address its precise functionality and to assess clinical efficacy and safety in different disease contexts. In this review, we outline clinical applications, efficacy, durability, and safety of FMT and provide a comprehensive assessment of its procedural and administration aspects. The clinical applications of FMT in children and cancer immunotherapy are also described. We focus on data from human studies in IBD in contrast with rCDI to delineate the putative mechanisms of this treatment in IBD as a model, including colonization resistance and functional restoration through bacterial engraftment, modulating effects of virome/phageome, gut metabolome and host interactions, and immunoregulatory actions of FMT. Furthermore, we comprehensively review omics technologies, metagenomic approaches, and bioinformatics pipelines to characterize complex microbial communities and discuss their limitations. FMT regulatory challenges, ethical considerations, and pharmacomicrobiomics are also highlighted to shed light on future development of tailored microbiome-based therapeutics.

Prospective and systematic screening for invasive aspergillosis in the ICU during the COVID-19 pandemic, a proof of principle for future pandemics.

The diagnostic performance of a prospective, systematic screening strategy for COVID-19 associated pulmonary aspergillosis (CAPA) during the COVID-19 pandemic was investigated. Patients with COVID-19 admitted to the ICU were screened for CAPA twice weekly by collection of tracheal aspirate (TA) for Aspergillus culture and PCR. Subsequently, bronchoalveolar lavage (BAL) sampling was performed in patients with positive screening results and clinical suspicion of infection. Patient data were collected from April 2020-February 2022. Patients were classified according to 2020 ECMM/ISHAM consensus criteria. In total, 126/370 (34%) patients were positive in screening and CAPA frequency was 52/370 (14%) (including 13 patients negative in screening). CAPA was confirmed in 32/43 (74%) screening positive patients who underwent BAL sampling. ICU mortality was 62% in patients with positive screening and confirmed CAPA, and 31% in CAPA cases who were screening negative. The sensitivity, specificity, positive and negative predictive value (PPV & NPV) of screening for CAPA were 0.71, 0.73, 0.27, and 0.95, respectively. The PPV was higher if screening was culture positive compared to PCR positive only, 0.42 and 0.12 respectively. CAPA was confirmed in 74% of screening positive patients, and culture of TA had a better diagnostic performance than PCR. Positive screening along with clinical manifestations appeared to be a good indication for BAL sampling since diagnosis of CAPA was confirmed in most of these patients. Prospective, systematic screening allowed to quickly gain insight into the epidemiology of fungal superinfections during the pandemic and could be applicable for future pandemics.

Genetic Determinants in MRSA Carriage and Their Association with Decolonization Outcome.

Methicillin-resistant Staphylococcus aureus (MRSA) colonization increases the risk of infection. Response to decolonization treatment is highly variable and determinants for successful decolonization or failure of eradication treatment are largely unknown. Insight into genetic predictors of eradication failure is potentially useful in clinical practice. The aim of this study was to explore genetic characteristics that are associated with MRSA decolonization failure. This cohort study was performed in a tertiary care hospital in the Netherlands. Patients with ≥ 1 positive MRSA culture from any site and with available whole -genome sequencing data of the MRSA isolate between 2017 and 2022 were included. Lineages, resistance, and virulence factors were stratified by MRSA decolonization outcome. In total, 56 patients were included: 12/56 (21%) with treatment failure and 44/56 (79%) with successful decolonization (with or without preceding treatment). A significant association was found between ciprofloxacin-resistant lineages and failure of eradication (OR 4.20, 95%CI 1.11-15.96, P = 0.04). Furthermore, livestock-associated MRSA and the major community-associated MRSA lineages ST6-t304 and ST8-t008 were associated with successful eradication treatment or spontaneous clearance. In conclusion, this explorative study showed a higher eradication failure rate in complicated MRSA carriers with ciprofloxacin-resistant MRSA lineages, which are predominantly healthcare-associated. Further studies are warranted to confirm the higher eradication failure risk of ciprofloxacin-resistant lineages, and identify the underlying mechanisms.

The ESCMID Study Group for Clostridioides difficile: History, Role, and Perspectives.

Clostridioides difficile (C. difficile) is a major nosocomial pathogen but is also increasingly recognised as an important diarrhoeal pathogen in the community, not always associated with antibiotics. The European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Study Group for C. difficile (ESGCD) is a group of clinicians, scientists, and others from many European countries and further afield, who share a common interest in C. difficile. The aims of the Study Group are centred around raising the profile of  C. difficile infection (CDI) in humans and animals, fostering collaboration amongst centres in different European countries and providing a forum for discussing and disseminating information. One of the principal aims of the Study Group is to raise awareness of C. difficile infections in Europe. ESGCD has a particular interest in the development and dissemination of European guidance on prevention, diagnosis, and treatment of CDI. This chapter will discuss the organisation of ESGCD within the ESCMID Study Group structure, the origins of the Study Group, the aims, and objectives of the group, and will highlight some of the past and present activities of ESGCD in relation to these.

Diagnostic Guidance for C. difficile Infections.

Diagnosis of Clostridioides difficile infection (CDI) can be challenging. First of all, there has been debate on which of the two reference assays, cell cytotoxicity neutralization assay (CCNA) or toxigenic culture (TC), should be considered the gold standard for CDI detection. Although the CCNA suffers most from suboptimal storage conditions and subsequent toxin degradation, TC is reported to falsely increase CDI detection rates as it cannot differentiate CDI patients from patients asymptomatically colonised by toxigenic C. difficile. Several rapid assays are available for CDI detection and fall into three broad categories: (1) enzyme immunoassays for glutamate dehydrogenase, (2) enzyme immunoassays or single-molecule array assays for toxins A/B and (3) nucleic acid amplification tests detecting toxin genes. All three categories have their own limitations, being suboptimal specificity and/or sensitivity or the inability to discern colonised patients from CDI patients. In light of these limitations, multi-step algorithmic testing has been advocated by international guidelines (IDSA/SHEA and ESCMID) in order to optimize diagnostic accuracy. As a result, a survey performed in 2018-2019 in Europe revealed that most of all hospital sites reported using more than one test to diagnose CDI. CDI incidence rates are also influenced by sample selection criteria, as several studies have shown that if not all unformed stool samples are tested for CDI, many cases may be missed due to an absence of clinical suspicion. Since methods for diagnosing CDI remain imperfect, there has been a growing interest in alternative testing strategies like faecal microbiota biomarkers, immune modulating interleukins, cytokines and imaging methods. At the moment, these alternative methods might play an adjunctive role, but they are not suitable to replace conventional CDI testing strategies.

Carriage of three plasmids in a single human clinical isolate of Clostridioides difficile.

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we provide the complete genome sequence of a human clinical isolate that carries three high-copy number plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile that is also compatible with the plasmid pCD630 that is found in many laboratory strains. Together, our data advance our understanding of C. difficile plasmid biology.

Impact of the COVID-19 pandemic on prevalence of highly resistant microorganisms in hospitalised patients in the Netherlands, March 2020 to August 2022.

BackgroundThe COVID-19 pandemic resulted in adaptation in infection control measures, increased patient transfer, high occupancy of intensive cares, downscaling of non-urgent medical procedures and decreased travelling.AimTo gain insight in the influence of these changes on antimicrobial resistance (AMR) prevalence in the Netherlands, a country with a low AMR prevalence, we estimated changes in demographics and prevalence of six highly resistant microorganisms (HRMO) in hospitalised patients in the Netherlands during COVID-19 waves (March-June 2020, October 2020-June 2021, October 2021-May 2022 and June-August 2022) and interwaves (July-September 2020 and July-September 2021) compared with pre-COVID-19 (March 2019-February 2020).MethodsWe investigated data on routine bacteriology cultures of hospitalised patients, obtained from 37 clinical microbiological laboratories participating in the national AMR surveillance. Demographic characteristics and HRMO prevalence were calculated as proportions and rates per 10,000 hospital admissions.ResultsAlthough no significant persistent changes in HRMO prevalence were detected, some relevant non-significant patterns were recognised in intensive care units. Compared with pre-COVID-19 we found a tendency towards higher prevalence of meticillin-resistant Staphylococcus aureus during waves and lower prevalence of multidrug-resistant Pseudomonas aeruginosa during interwaves. Additionally, during the first three waves, we observed significantly higher proportions and rates of cultures with Enterococcus faecium (pooled 10% vs 6% and 240 vs 120 per 10,000 admissions) and coagulase-negative Staphylococci (pooled 21% vs 14% and 500 vs 252 per 10,000 admissions) compared with pre-COVID-19.ConclusionWe observed no substantial changes in HRMO prevalence in hospitalised patients during the COVID-19 pandemic.

Community-onset Clostridioides difficile infection in south Serbia.

BACKGROUND: Data from the past decade indicates that Clostridioides difficile infection (CDI) is not only a nosocomial infection but is also increasingly recognized as a disease in the community. OBJECTIVE: We aimed to study community-onset (CO) CDI in the various age groups in south Serbia with its clinical characteristics, risk factors and microbiological characterization. METHODS: The study group included 93 patients with CO-CDI (median age 62). The control group consisted of 186 patients with community-onset diarrhea and stool samples negative tested for CDI. RESULTS: Of all CDI cases diagnosed with a community onset, 74.19% had a previous contact with a healthcare facility in the previous 12 weeks, but 34.40% have no record on hospitalization in the previous 12 months. Using a multivariate statistical regression model, the following risk factors for CO-CDI development were found; antacid usage (OR = 0.267, 95%C.I.:0.10-0.291, p < 0.01), chronic kidney disease (OR = 0.234, 95%C.I.:0.10-0.51, p < 0.01) and antibiotic use during the prior 2 months (OR = 0.061, 95%C.I.:0.02-0.17, p < 0.01), especially tetracycline's (OR = 0.146, 95% C.I.:0.07-0.22, p < 0.01) and cephalosporin's (OR = 0.110, 95%C.I.:0.14-0.42, p < 0.01). The most common ribotypes (RTs) detected in patients with CO-CDI were RT001 (32.3%) and RT027 (24.7%). All tested toxin producing C. difficile isolates were sensitive to metronidazole, vancomycin and tigecycline. A high rate of resistance to moxifloxacin (73.11%) and rifampicin (23.65%) was found. CONCLUSION: Patients with CO-CDI had frequently contact with healthcare facility in the previous 12 weeks. Restriction of antacid usage and of high-risk antibiotics in the community may help reduce the incidence of CO-CDI.

Incidence of healthcare-associated Clostridioides difficile infection in a quaternary referral university hospital in Brazil.

Clostridioides difficile infection (CDI) is an important cause of diarrhea in hospitals worldwide. The incidence of CDI in Latin America has not yet been standardized. To fill this gap, the present study performed a daily active surveillance, for three months, between April to July of 2021, at a quaternary referral university hospital in Brazil. The incidence density was 9.2 cases per 10,000 patient-days. Cases were associated mostly with ribotypes 014 and 106 (44% and 22%, respectively). Ribotype 027 was not identified. The findings strongly reinforce the need for broad epidemiological studies on the incidence of CDI in Brazilian hospitals to increase the understanding, prevention, and treatment of this infection.

Sequence-Based Identification of Metronidazole-Resistant Clostridioides difficile Isolates.

The plasmid pCD-METRO confers metronidazole resistance in Clostridioides difficile. We showed high sequence similarity among pCD-METRO plasmids from different isolates and identified pCD-METRO and associated metronidazole-resistant isolates in clinical and veterinary reservoirs in the Americas. We recommend using PCR or genomic assays to detect pCD-METRO in metronidazole-resistant C. difficile.

Fecal Microbiota Transplantation Influences Procarcinogenic Escherichia coli in Recipient Recurrent Clostridioides difficile Patients.

BACKGROUND & AIMS: Patients with multiple recurrent Clostridioides difficile infection (rCDI) have a disturbed gut microbiota that can be restored by fecal microbiota transplantation (FMT). Despite extensive screening, healthy feces donors may carry bacteria in their intestinal tract that could have long-term health effects, such as potentially procarcinogenic polyketide synthase-positive (pks+) Escherichia coli. Here, we aim to determine whether the pks abundance and persistence of pks+E coli is influenced by pks status of the donor feces. METHODS: In a cohort of 49 patients with rCDI treated with FMT and matching donor samples-the largest cohort of its kind, to our knowledge-we retrospectively screened fecal metagenomes for pks+E coli and compared the presence of pks in patients before and after treatment and to their respective donors. RESULTS: The pks island was more prevalent (P = .026) and abundant (P < .001) in patients with rCDI (pre-FMT, 27 of 49 [55%]; median, 0.46 reads per kilobase per million [RPKM] pks) than in healthy donors (3 of 8 donors [37.5%], 11 of 38 samples [29%]; median, 0.01 RPKM pks). The pks status of patients post-FMT depended on the pks status of the donor suspension with which the patient was treated (P = .046). Particularly, persistence (8 of 9 cases) or clearance (13 of 18) of pks+E coli in pks+ patients was correlated to pks in the donor (P = .004). CONCLUSIONS: We conclude that FMT contributes to pks+E coli persistence or eradication in patients with rCDI but that donor-to-patient transmission of pks+E coli is unlikely.

Faecal microbiota replacement to eradicate antimicrobial resistant bacteria in the intestinal tract - a systematic review.

PURPOSE OF REVIEW: Antimicrobial resistance is a rising threat to global health and is associated with increased mortality. Intestinal colonisation with multidrug-resistant organisms (MDRO) can precede invasive infection and facilitates spread within communities and hospitals. Novel decolonisation strategies, such as faecal microbiota transplantation (FMT), are being explored. The purpose of this review is to provide an update on how the field of FMT for MDRO decolonisation has developed during the past year and to assess the efficacy of FMT for intestinal MDRO decolonisation. RECENT FINDINGS: Since 2020, seven highly heterogenous, small, nonrandomised cohort studies and five case reports have been published. In line with previous literature, decolonisation rates ranged from 20 to 90% between studies and were slightly higher for carbapenem-resistant Enterobacteriaceae than vancomycin-resistant Enterococcus. Despite moderate decolonisation rates in two studies, a reduction in MDRO bloodstream and urinary tract infections was observed. SUMMARY AND IMPLICATIONS: Although a number of smaller cohort studies show some effect of FMT for MDRO decolonisation, questions remain regarding the true efficacy of FMT (taking spontaneous decolonisation into account), the optimal route of administration, the role of antibiotics pre and post-FMT and the efficacy in different patient populations. The observed decrease in MDRO infections post-FMT warrants further research.

Faecal carriage of Clostridioides difficile is low among veterinary healthcare workers in the Netherlands.

Veterinary healthcare workers are in close contact with many different animals and might be at an increased risk of acquiring Clostridioides difficile. In this cross-sectional study, we assessed the prevalence and risk factors of C. difficile carriage in Dutch veterinary healthcare workers. Participants provided a faecal sample and filled out a questionnaire covering potential risk factors for C. difficile carriage. C. difficile culture positive isolates were polymerase chain reaction (PCR) ribotyped and the presence of toxin genes tcdA, tcdB and cdtA/cdtB was determined. Eleven of 482 [2.3%; 95% confidence interval (CI) 1.3-4.0] veterinary healthcare workers were carriers of C. difficile. Three persons carried C. difficile ribotype 078 (0.6%; 95% CI 0.2-1.8). Risk factors for carriage were health/medication and hygiene related, including poor hand hygiene after patient (animal) contact, and did not include occupational contact with certain animal species. In conclusion, the prevalence of C. difficile carriage in veterinary healthcare workers was low and no indications were found that working in veterinary care is a risk for C. difficile carriage.

An unusual outbreak in the Netherlands: community-onset impetigo caused by a meticillin-resistant Staphylococcus aureus with additional resistance to fusidic acid, June 2018 to January 2020.

In this retrospective observational study, we analysed a community outbreak of impetigo with meticillin-resistant Staphylococcus aureus (MRSA), with additional resistance to fusidic acid (first-line treatment). The outbreak occurred between June 2018 and January 2020 in the eastern part of the Netherlands with an epidemiological link to three cases from the north-western part. Forty nine impetigo cases and eight carrier cases were identified, including 47 children. All but one impetigo case had community-onset of symptoms. Pharmacy prescription data for topical mupirocin and fusidic acid and GP questionnaires suggested an underestimated outbreak size. The 57 outbreak isolates were identified by the Dutch MRSA surveillance as MLVA-type MT4627 and sequence type 121, previously reported only once in 2014. Next-generation sequencing revealed they contained a fusidic acid resistance gene, exfoliative toxin genes and an epidermal cell differentiation inhibitor gene. Whole-genome multilocus sequence typing revealed genetic clustering of all 19 sequenced isolates from the outbreak region and isolates from the three north-western cases. The allelic distances between these Dutch isolates and international isolates were high. This outbreak shows the appearance of community-onset MRSA strains with additional drug resistance and virulence factors in a country with a low prevalence of antimicrobial resistance.

Multidrug-resistant organisms in patients from Ukraine in the Netherlands, March to August 2022.

Since March 2022, there has been an emergence of multidrug-resistant organisms (MDRO) in the Netherlands in patients originating from Ukraine (58 patients, 75 isolates). For about half of these patients, recent hospitalisation in Ukraine was reported. Genomic surveillance revealed that the majority of the MDRO represent globally spread epidemic lineages and that 60% contain New Delhi metallo-ß-lactamase (NDM) genes. Professionals should be aware of an increase in such MDRO associated with migration and medical evacuation of people from Ukraine.

In vitro anti-clostridial action and potential of the spice herbs essential oils to prevent biofilm formation of hypervirulent Clostridioides difficile strains isolated from hospitalized patients with CDI.

BACKGROUND: Clostridioides difficile is the most common causative agent of antibiotic-acquired diarrhea in hospitalized patients associated with substantial morbidity and mortality. The global epidemic of CDI (Clostridioides difficile infection) began in the early 20th century with the emergence of the hypervirulent and resistant ribotype 027 strains, and requires an urgent search for new therapeutic agents. OBJECTIVE: The aim of this study is to investigate the antibacterial activity of the three essential oils isolated from spice herbs (wild oregano, garlic and black pepper) against C. difficile clinical isolates belonging to 6 different PCR ribotypes and their potential inhibitory effect on the biofilm production in in vitro conditions. RESULTS: Wild oregano essential oil showed strong inhibitory activity in concentrations 0.02-1.25 mg/mL and bactericidal activity in concentrations from 0.08 to 10 mg/mL. Garlic essential oil was effective in the concentration range of 0.02-40 mg/mL, and 0.16 - > 40 mg/mL. MIC and MBC for black pepper oil ranged from 0.04 to 40 mg/mL, and 0.08 - > 40 mg/mL, respectively. All the tested oils reduced in vitro biofilm production, with the best activity of oregano oil. CONCLUSION: Essential oils of wild oregano, black pepper and garlic are candidates for adjunctive therapeutics in the treatment of CDI. Oregano oil should certainly be preferred due to the lack of selectivity of action in relation to the ribotype, the strength of the produced biofilm and/or antibiotic-susceptibility patterns.

Opportunities and Challenges in Development of Live Biotherapeutic Products to Fight Infections.

Treatment of bacterial infections with broad-spectrum antibiotics is a strategy severely limited by the decreased ability of the perturbed resident microbiota to control expansion of antibiotic-resistant pathogens. Live biotherapeutic products (LBPs) could provide an alternative to antibiotics in infection control by restoring gut colonization resistance and controlling expansion of resistant strains, an important therapeutic need not being addressed with existing anti-infective drug modalities. We review opportunities and challenges in developing LBPs for multidrug-resistant organisms colonization and infection control, with a focus on commercial fecal microbiota transplantation-like products and defined bacterial consortia, and spanning considerations related to availability of models for rational drug candidate selection and dose regimen selection, good manufacturing practice, intellectual property, and commercial viability.

Haem is crucial for medium-dependent metronidazole resistance in clinical isolates of Clostridioides difficile.

BACKGROUND: Until recently, metronidazole was the first-line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. OBJECTIVES: To identify factors that contribute to plasmid-independent metronidazole resistance of C. difficile. METHODS: Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile using a combination of antimicrobial susceptibility testing on different solid agar media and WGS of selected isolates. RESULTS: We find that nearly all isolates demonstrate a haem-dependent increase in the MIC of metronidazole, which in some cases leads to isolates qualifying as resistant (MIC >2 mg/L). Moreover, we find an SNP in the haem-responsive gene hsmA, which defines a metronidazole-resistant lineage of PCR ribotype 010/MLST ST15 isolates that also includes pCD-METRO-containing strains. CONCLUSIONS: Our data demonstrate that haem is crucial for medium-dependent metronidazole resistance in C. difficile.

The vaginal microbiota in the course of bacterial vaginosis treatment.

Bacterial vaginosis (BV) is perceived as a condition of disrupted vaginal microbiota, but remains of unknown aetiology. In this study, vaginal microbiota composition was determined in twenty-one women with BV, before and after treatment with metronidazole or clindamycin. Microbiota composition varied greatly between women and defining a (un)healthy vaginal microbiota state remains elusive, challenging BV diagnosis and treatment. While relative abundance of Lactobacillus increased after antibiotic treatment in two-third of women, its abundance was not associated with treatment outcome. Instead, remaining complaints of abnormal vaginal discharge were more common after metronidazole treatment and associated with increased relative abundance of Ureaplasma.

Systematic screening for COVID-19 associated invasive aspergillosis in ICU patients by culture and PCR on tracheal aspirate.

BACKGROUND: A high prevalence of COVID-19 associated pulmonary aspergillosis (CAPA) has been reported, though histopathological evidence is frequently lacking. To assess the clinical significance of Aspergillus species in respiratory samples of mechanically ventilated COVID-19 patients, we implemented routine screening for Aspergillus in tracheal aspirate (TA). PATIENTS/METHODS: From all adult COVID-19 patients admitted to the intensive care unit (ICU), TA samples were collected twice a week for Aspergillus screening by PCR and or culture. Bronchoalveolar lavage (BAL) sampling was performed in patients with a positive screening result if possible. Clinical information was obtained from the electronic patient record and patients were categorised according to the recently published consensus case definition for CAPA. RESULTS: Our study population consisted of 63 predominantly (73%) male patients, with a median age of 62 years and total median ICU stay of 18 days. Aspergillus species were present in TA screening samples from 15 patients (24%), and probable CAPA was diagnosed in 11 (17%) patients. Triazole resistance was detected in one patient (14%). Concordance between TA and BAL was 86%, and all TA culture positives were confirmed in BAL. We were able to withhold treatment in three of fifteen patients with positive screening (20%) but negative BAL results. CONCLUSIONS: Positive culture, molecular detection and or antigen detection of Aspergillus species do not equal infection. Until we understand the clinical relevance of Aspergillus species detected in respiratory samples of COVID-19 patients, minimal-invasive screening by TA is a feasible method to monitor patients. Positive screening results should be an indication to perform a BAL to rule out upper airway colonisation.