As antimicrobial resistance continues to rise and the development of new antimicrobials lags, we face an urgent threat where routine infections could become life-threatening. Armed conflicts, like the Russian war on Ukraine, intensify this crisis by accelerating the emergence and spread of resistant organisms across regions and borders. Phage Support Ukraine (Phage Sp UKR) is a collaborative effort to provide phages targeting circulating pathogens in Ukraine for delivery of personalized phage therapies. Within this context, we received multidrug-resistant bacterial isolates belonging to a number of different species. We here focus on Klebsiella pneumoniae , the most concerning pathogen with regard to prevalence, virulence and antibiotic resistance. Among the K. pneumoniae isolates, we identified sequence types (ST) 39, 101, 147, 307 and 395, as well as a predominating clonal group—CG10146 (ST23 KL57), first detected in Moscow—carrying a hybrid virulence-resistance plasmid encoding NDM-1 and aerobactin. Over two years (2023–2025), this clonal group expanded and acquired pan-resistance. When tested in virulence assays, only one isolate (ST395) displayed hypervirulence in a Galleria mellonella though not in a mouse infection model. Several phages isolated from Ukrainian sewage were able to lyse these K. pneumoniae strains, including strains obtained from soldiers displaced in Belgium, Germany, and Latvia. Bacterial resistance was observed during in vitro testing. However, some phage-resistant isolates had mutations in virulence factors, including one that completely lost its hybrid plasmid, resulting in restored antibiotic susceptibility. Strategies like Phage Sp UKR are essential to prevent the selection, persistence, and global spread of these MDR clones. ### Competing Interest Statement The authors have declared no competing interest.

OBJECTIVES:Multidrug-resistant (MDR) Klebsiella pneumoniae is an increasingly important cause of recurrent urinary tract infections (UTIs). As bacteriophage therapy represents a promising alternative, we aimed to isolate and characterize bacteriophages targeting a clinical MDR K. pneumoniae strain causing recurrent UTI and evaluate their activity under urinary conditions. METHODS:Three bacteriophages targeting an ESBL-producing K. pneumoniae clinical isolate were obtained and characterized by genome sequencing, electron microscopy, stability assays, one-step growth curves, and host-range analysis across 79 UTI isolates. Phage activity was quantified in LB medium and human urine. RESULTS:Three lytic phages (EDIRA083, EDIRA088, and EDIRA092) belonging to distinct genera were identified. Absence of lysogeny-associated, virulence, or antibiotic-resistance genes was confirmed. Capsule, LPS and maltoporin LamB were identified as receptors for EDIRA083 and EDIRA092. Host-range analysis revealed narrow activity for EDIRA083 and EDIRA088, whereas EDIRA092 infected 29% of the panel strains. In liquid phage infection assays, overall lytic activity was consistently higher and more sustained in human urine than in LB. CONCLUSIONS:These results identify three genetically distinct lytic phages targeting MDR K. pneumoniae. Their activity in urine supports further evaluation of these phages as candidates for therapeutic development against MDR Klebsiella UTI.

Clostridioides difficile (CD) infections are defined by toxins A (TcdA) and B (TcdB) along with the binary toxin (CDT). The emergence of the 'hypervirulent' (Hv) strain PR 027, along with PR 176 and 181, two decades ago, reshaped CD infection epidemiology in Europe. This study assessed MALDI-TOF mass spectrometry (MALDI-TOF MS) combined with machine learning (ML) and Deep Learning (DL) to identify toxigenic strains (producing TcdA, TcdB with or without CDT) and Hv strains. In total, 201 CD strains were analysed, comprising 151 toxigenic (24 ToxA(+)B(+)CDT(+), 22 ToxA(+)B(+)CDT(+) Hv(+) and 105 ToxA(+)B(+)CDT(-)) and 50 non-toxigenic (ToxA(-)B(-)) strains. The DL-based classifier exhibited a 0.95 negative predictive value for excluding ToxA(-)B(-) strains, showcasing accuracy in identifying this strain category. Sensitivity in correctly identifying ToxA(+)B(+)CDT(-) strains ranged from 0.68 to 0.91. Additionally, all classifiers consistently demonstrated high specificity (>0.96) in detecting ToxA(+)B(+)CDT(+) strains. The classifiers' performances for Hv strain detection were linked to high specificity (>= 0.96). This study highlights MALDI-TOF MS enhanced by ML techniques as a rapid and cost-effective tool for identifying CD strain virulence factors. Our results brought a proof-of-concept concerning the ability of MALDI-TOF MS coupled with ML techniques to detect virulence factor and potentially improve the outbreak's management.

The present study describes the first isolation of a recently described Campylobacter species, Campylobacter vicugnae, in humans. The isolates were recovered by two independent French laboratories in 2020 and 2022 from a man and a woman suffering from gastroenteritis. Biochemical and growth characteristics, and electron microscopy for these two strains indicated that they belong to Campylobacter genus. 16S rDNA and GyrA-based phylogeny, as well as average nucleotide identity and in silico DNA-DNA Hybridization analyses revealed that both strains belong to the Campylobacter vicugnae species. Both isolates possess a complete cytolethal distending toxin (CDT) locus with cdtA, cdtB, and cdtC, and features of CDT activity were demonstrated in vitro with Caco-2 intestinal epithelial cells. Our data suggest that these two isolates of C. vicugnae were associated with gastroenteritis in humans and induced major cytopathogenic effects in vitro. C. vicugnae is likely to be a novel human pathogen, with a source of foodborne infection that needs to be determined.IMPORTANCECampylobacter species that display toxicity features are a worldwide public health issue. In clinical contexts, it is crucial to identify which isolate could be an urgent threat to a patient. Actual and widely used laboratory methods such as mass spectrometry or PCR may be flawed in the field of species identification. In contrast, the present study shows that next-generation sequencing allows to precisely identify isolates to species level that may have been omitted otherwise. Moreover, it helps to identify emerging species before they become a threat to human health. Recovery of a new Campylobacter species in human sample, such as the new species “Campylobacter vicugnae,” is an important step for the identification of emerging pathogens posing threat to global health.

Preparing fecal microbiota transplants immediately after donation is resource-intensive, and a proportion are destroyed following abnormal screening results. We retrospectively compared two processes, frozen fecal preparation (FFP) and fresh native frozen preparation (FNFP), for clinical efficacy in the treatment of recurrent Clostridioides difficile infection (rCDI). FFP and FNFP were similarly effective with clinical success rates of 76.7% and 86.7% (P = 0.32), respectively. FNFP is an efficient procedure that saves resources while maintaining clinical efficacy in rCDI.

Antimicrobial resistance is a major threat to human and animal health and accounted for up to 4.5 million deaths worldwide in 2019. Asymptomatic colonization of the digestive tract by multidrug resistant (multi-resistant) bacteria such as extended-spectrum beta-lactamase-, or carbapenemase- producing Enterobacterales is (i) a risk factor for infection by these multi-resistant bacteria, (ii) a risk factor of dissemination of these multi-resistant bacteria among patients and in the community, and (iii) allows the exchange of resistance genes between bacteria. Hence, decolonization or reduction of the gastrointestinal tract colonization of these multi-resistant bacteria needs to be urgently explored. Developing new non-antibiotic strategies to limit or eradicate multi-resistant bacteria carriage without globally disrupting the microbiota is considered a priority to fight against antibiotic resistance. Probiotics or Fecal Microbiota Transplantation are alternative strategies to antibiotics that have been considered to decolonize intestinal tract from MDR bacteria but there is currently no evidence demonstrating their efficacy. Lytic bacteriophages are viruses that kill bacteria and therefore could be considered as a promising strategy to combat antibiotic resistance. Successful decolonization by bacteriophages has already been observed clinically. Here, we discuss the current alternative strategies considered to decolonize the digestive tract of multidrug resistant bacteria, briefly describing probiotics and fecal microbiota transplantation approaches, and then detail the in vivo and in vitro studies using bacteriophages, while discussing their limits regarding the animal models used, the characteristics of phages used and their activity in regards of the gut anatomy.

CLOSTRIDIOIDES DIFFICILE:UPDATED RECOMMENDATIONS Clostridioides difficile is a spore-forming anaerobic enteropathogen responsible for a wide spectrum of clinical features ranging from mild uncomplicated diarrhoea to severe debilitating disease, toxic megacolon, or even perforation and sometimes death. Risk factors for CDI include age >65 years, previous hospitalization and recent antibiotic therapy. Main virulence factors of C. difficile are toxins A (TcdA) and B (TcdB). The emergence and dissemination of a new hypervirulent strain (027/NAP/BI) in 2005 has stimulated clinical and basic research on C. difficile. Major advances have been made regarding the CDI epidemiology (better recognition of community acquired CDI), diagnosis (molecular tests) and therapy (new drugs such as fidaxomicin, bezlotoxumab, and fecal microbiota transplantation) aspects. These advances have allowed the updating of management recommendations, under the aegis of the European Society of Clinical Microbiology and Infectious Diseases (ESCMID). Antibiotic treatment of CDI depends on both the severity of the infection, and the risk/number of recurrences. Prevention of CDI requires an antimicrobial stewardship policy and the implementation of contact precautions for the infected patients.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is commonly used by clinical microbiology laboratories to identify pathogens, despite some limitations of the technique. The Enterobacter cloacae complex (ECC) taxonomy has recently been expanded, leading to uncertain identification of some species within the ECC when commercial MALDI-TOF MS is used. This technique is especially unsuited in the case of E. hormaechei, the main species responsible for infections and one of the most prone, within the ECC, to acquire antibiotic resistance. Hence, rapid and reliable identification at the species level could improve patient management. Here, we evaluated the performance of the Bruker Microflex MALDI-TOF MS instrument to identify ECC isolates using two databases and algorithms in comparison to the hsp60 gene sequencing reference method: the Bruker database included in the MALDI Biotyper software and an extensive online database coupled to an original Mass Spectrometric Identification (MSI) algorithm. Among a panel of 94 ECC isolates tested in triplicate, the online database coupled to MSI software allowed the highest rate of identification at the species level (92%) compared to the MALDI Biotyper database (25%), especially for the species E. hormaechei (97% versus 20%). We show that by creating a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software, we were able to substantially improve the identification of the E. cloacae complex members, with only 8% of isolates misidentified at the species level. This online database is available through a free online MSI application (https://msi.happy-dev.fr/). IMPORTANCE Creation of a database of MALDI-TOF reference spectral profiles with a high number of representatives associated with the performant MSI software enables substantial improvement in identification of E. cloacae complex members. Moreover, this online database is available through a free online MSI application (https://msi.happy-dev.fr/).

Reported rates of C. difficile infection (CDI) have increased in many settings; however, these can be affected by factors including testing density (test-density) and diagnostic methods. We aimed to describe the impact of multiple factors on CDI rates. Hospitals (n = 182) across five countries (France, Germany, Italy, Spain, and UK) provided data on; size and type of institution, CDI testing methodology, number of tests/month and patient-bed-days (pbds)/month over one year. Incidence rates were compared between countries, different sized institutions, types of institutions and testing method. After univariate analyses, the highest CDI rates were observed in Italy (average 11.8/10,000pbds/hospital/month), acute/primary hospitals (12.3/10,000pbds/hospital/month), small hospitals (16.7/10,000pbds/hospital/month), and hospitals using methods that do not detect toxin (NO-TOXIN) (e.g. GDH/NAAT or standalone NAAT) (10.7/10,000pbds/hospital/month). After adjusting for test-density, highest incidence rates were still in Italy, acute/primary hospitals and those using NO-TOXIN. The relative rate in long-term healthcare facilities (LTHCFs) increased, but size of institution no longer influenced the CDI rate. Test-density appears to have the largest effect on reported CDI rates. NO-TOXIN testing still influences CDI rates, even after adjusting for test-density, which is consistent with tests that 'overcall' true CDI. Low test-density can mask the true burden of CDI, e.g. in LTHCFs, highlighting the importance of good quality surveillance. (C) 2020 Elsevier Ltd. All rights reserved.

Faropenem is an oral penem drug with activity against Gram-positive and Gram-negative bacteria, including CTX-M-15-type extended spectrum beta-lactamase (ESBL)-producing Enterobacteriales and anaerobic bacteria. As there are structural similarities, there is concern for the development of carbapenem cross-resistance; however, there are no studies confirming this. This study examined whether in vitro development of faropenem resistance in Escherichia coli isolates would result in cross-resistance to carbapenems.Four well-characterized E. coli isolates from the US Centers for Disease Control and Prevention antibiotic resistance isolate bank were utilized. Three isolates (NSF1, NSF2 and NSF3) are ESBL producers (CTX-M-15) and one (NSF4) is pan-susceptible. Faropenem minimum inhibitory concentrations (MICs) were determined and resistance was induced by serial passaging in increasing concentrations of faropenem. Susceptibility to carbapenems was determined and whole-genome sequencing (WGS) was performed to identify the underlying genetic mechanism leading to carbapenem resistance.Faropenem MIC increased from 1 mg/L to 64 mg/L within 10 days for NSF2 and NSF4 isolates, and from 2 mg/L to 64 mg/L within 7 days for NSF1 and NSF3 isolates. Reduced carbapenem susceptibility (ertapenem MIC ≥8 mg/L, doripenem/meropenem ≥2 mg/L and imipenem ≥1 mg/L) developed among three CTX-M-15-producing isolates that were faropenem-resistant, but not in NSF4 isolate that lacked ESBL enzyme. WGS analysis revealed non-synonymous changes in the ompC gene among three CTX-M-15-producing isolates, and a single nucleotide polymorphism (SNP) in the envZ gene in NSF4 isolate.Induced resistance to faropenem causes cross-resistance to carbapenems among E. coli isolates containing CTX-M-15-type ESBL enzymes.

Pathogenesis of Clostridium difficile has been linked to production of toxins, including the large toxins A and B as well as the binary toxin CDT. Until recently, toxin A was only found in combination in clinical strains with the toxin B, unlike toxin B or CDT, which were found alone in toxigenic variants. New toxigenic variants of C. difficile detected in our laboratory from patients with diarrhoea or severe colitis, including a variant producing only toxin A, were tested for virulence in the hamster model, which displays the clinical features of C. difficile disease. Hamsters infected with a strain producing only toxin B induced similar clinical signs, time to death from infection and histologic damage compared to the hypervirulent strain 027. No mortality or clinical signs of infection but caecal histologic damage was found with the variant producing only toxin A. The C. difficile variant strain producing only CDT was able to kill one hamster out of seven; nevertheless, the surviving animals had few alteration of the caecum.

Objectives: Reported rates of community-acquired Clostridium difficile infections (CDIs) have been increasing. However, the true burden of the disease in general practice is unknown in France. Our objective was to determine the incidence of toxigenic C. difficile carriage and the percentage of stool samples prescribed by general practitioners (GPs) which contained free C. difficile toxins. Methods: During an 11-month period, all stool samples submitted for any enteric pathogen detection to 15 different private laboratories in Paris and the surrounding areas were tested for C. difficile, irrespective of the GPs' request. A clinical questionnaire was completed for each patient. Stool samples were screened using a rapid simultaneous glutamate dehydrogenase and toxins A/B detection test: any positive result (glutamate dehydrogenase or toxin) was further confirmed by the stool cytotoxicity assay (CTA) on MRC-5 cells and by toxigenic culture (TC) at a central laboratory. The C. difficile isolates were characterized by PCR ribotyping. Results: A total of 2541 patients (1295 female, 1246 male) were included. The incidences of patients with a positive toxigenic culture and a positive CTA were 3.27% (95% CI 2.61%-4.03%) and 1.81% (95% CI 1.33%-2.41%), respectively. GPs requested C. difficile testing in only 12.93% of the stool samples, detecting 52.30% of all TC-positive patients. The 83 toxigenic C. difficile strains belonged to 36 different PCR ribotypes. Conclusions: Toxigenic C. difficile carriage is frequent in general practice but remains under-recognized. It may affect young patients without previous antimicrobial therapy or hospitalization. (C) 2019 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Background Premature neonates (PN) present multiple risk factors for high frequencies and high levels of colonization by C. difficile, yet data is missing about this specific pediatric population. Here, we investigated PN C. difficile carriage and colonization dynamics, analyzed the impact of perinatal determinants on colonization, and characterized the isolates. Methods A one year longitudinal monocentric prospective cohort study was performed on 121 PN. C. difficile strains isolated from fecal samples on selective medium were identified and characterized by PCR (tpi housekeeping gene; tcdA and tcdB, and binary toxin genes), capillary gel-based electrophoresis PCR-ribotyping, and Multi-Locus Variable-number tandem-repeat Analysis (MLVA). Results Of the 379 samples analyzed, 199 (52%) were C. difficile culture positive with the mean levels of C. difficile colonization decreasing significantly (P = .027) over time. During hospitalization, C. difficile colonization frequency increased up to 61% with 95% of the strains belonging to both non-toxigenic PCR-ribotypes (RTs) FR082 (35%) and 032 (60%). After hospital discharge, if a higher diversity in RTs was observed, RTs FR082 and 032 remained predominant (respectively 40% and 28%). MLVA showed clonal relationship within each FR082 and 032 RTs. Ten toxigenic strains (5%) were isolated, all tcdA+/tcdB+ except for one tcdA-/tcdB+, and all being acquired after hospitalization. At 1 week, the only factors found to be linked with a higher frequency of C. difficile colonization were a higher gestational age (P = 0.006) and a higher birth weight (P = 0.016). Conclusion The dynamics of C. difficile colonization in PN followed a specific pattern. C. difficile colonization rapidly occurred after birth with a low diversity of non-toxigenic RTs. After hospitalization, non-toxigenic RTs diversity increased. Sporadic carriage of toxigenic strains was observed after hospitalization.

Historically, Clostridioides difficile ( Clostridium difficile ) has been associated with life-threatening diarrhea in hospitalized patients. Increasing rates of C. difficile infection (CDI) in the community suggest exposure to C. difficile reservoirs outside the hospital, including animals, the environment, or food. C. difficile sequence type 11 (ST11) is known to infect/colonize livestock worldwide and comprises multiple ribotypes, many of which cause disease in humans, suggesting CDI may be a zoonosis. Using high-resolution genomics, we investigated the evolution and zoonotic potential of ST11 and a new closely related ST258 lineage sourced from diverse origins. We found multiple intra- and interspecies clonal transmission events in all ribotype sublineages. Clones were spread across multiple continents, often without any health care association, indicative of zoonotic/anthroponotic long-range dissemination in the community. ST11 possesses a massive pan-genome and numerous clinically important antimicrobial resistance elements and prophages, which likely contribute to the success of this globally disseminated lineage of One Health importance.

Clostridium difficile est un bacille à Gram positif anaérobie et sporulé, reconnu comme le principal agent de diarrhées nosocomiales de l’adulte. Cette bactérie est responsable de 15 à 25 % des diarrhées post-antibiotique et de plus de 95 % des colites pseudomembraneuses. Les principaux facteurs de risques d’infection sont l’âge (? 65 ans), l’administration d’antibiotiques (principalement céphalosporines de troisième génération, amoxicilline associée à l’acide clavulanique, clindamycine et fluoroquinolones) et l’hospitalisation. Le diagnostic d’infection à C. difficile (ICD) repose sur les signes cliniques et les tests de laboratoire confirmant la présence d’une souche toxinogène ou de toxines dans les selles. Dans un contexte de récente augmentation de l’incidence et de la gravité des ICD, un diagnostic fiable est essentiel pour optimiser le traitement et la prévention.

The objective of this study was to evaluate the Amplidiag C. difficile+027 (R) assay, a new molecular method that detects toxin B gene in stool samples and identifies the hypervirulent 027 strain, to diagnose Clostridium difficile infections. The assay was compared to the reference method i.e. toxigenic culture. Amplidiag C. difficile+027 (R) assay was prospectively evaluated from 309 diarrheal stool specimens of patients suspected of C. difficile infection. Forty-five (14.6%) stools were positive by toxigenic culture and 11 (3.6%) stools gave discordant results with the molecular method. PR027 was not recovered during the study. After resolving the discrepant results, the sensitivity, specificity, positive and negative predictive values of Amplidiag C. difficile+027 (R) assay were 91.1% [CI 95% 77.9-97.1], 99.6% [CI 95% 97.6-100], 97.6% [CI 95% 85.9-99.9] and 98.5% [CI 95% 96-99.5], respectively compared to toxigenic culture. This assay is sensitive compared to the toxigenic culture. (C) 2018 Elsevier Ltd. All rights reserved.

Clostridium difficile infection (CDI) produces a variety of clinical presentations ranging from mild diarrhea to severe infection with fulminant colitis, septic shock, and death. CDI puts a heavy burden on healthcare systems due to increased morbidity and mortality, and higher costs. We evaluated the clinical impact of CDI in terms of complications and mortality in a French university hospital compared with patients with diarrhea unrelated to CDI. A 3-year prospective, observational, cohort study was conducted in a French university hospital. Inpatients aged 18 years or older with CDI-suspected diarrhea were eligible to participate in the study and were followed for up to 60 days after CDI testing. Among the 945 patients with diarrhea included, 233 had confirmed CDI. Overall, 106 patients (11.2%) developed at least one of the following complications: colectomy, colitis, ileitis/rectitis, ileus, intestinal perforation, megacolon, multiorgan failure, pancolitis, peritonitis, pseudomembranous colitis, renal failure, and sepsis/septic shock. The complication rate was significantly higher in patients with diarrhea related to C. difficile than in non-CDI patients (26.6% vs 6.2%, P < 0.001). At day 60, 137 (14.5%) patients had died, with 37 deaths among the CDI group (15.9%). Death was attributable to CDI in 15 patients (6.4%). Complications are more frequent among CDI cases than in patients with diarrhea not related to C. difficile. Assessment of CDI is necessary to ensure allocation of sufficient resources to CDI prevention.

Scope: Clostridium difficile infection (CDI) is the most important infective cause of healthcare-associated diarrhoea in high income countries and one of the most important healthcare-associated pathogens in both Europe and the United States. It is associated with high morbidity and mortality resulting in both societal and financial burden. A significant proportion of this burden is potentially preventable by a combination of targeted infection prevention and control measures and antimicrobial stewardship. The aim of this guidance document is to provide an update on recommendations for prevention of CDI in acute care settings to provide guidance to those responsible for institutional infection prevention and control programmes. Methods: An expert group was set up by the European society of clinical microbiology and infectious diseases (ESCMID) Study Group for C. difficile (ESGCD), which performed a systematic review of the literature on prevention of CDI in adults hospitalized in acute care settings and derived respective recommendations according to the GRADE approach. Recommendations are stratified for both outbreak and endemic settings. Questions addressed by the guideline and recommendations: This guidance document provides thirty-six statements on strategies to prevent CDI in acute care settings, including 18 strong recommendations. No recommendation was provided for three questions. (c) 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Calprotectin and lactoferrin are released by the gastrointestinal tract in response to infection and mucosal inflammation. Our objective was to assess the usefulness of quantifying faecal lactoferrin and calprotectin concentrations in Clostridium difficile infection (CDI) patients with or without free toxins in the stools. We conducted a single-centre 22-month case–control study. Patients with a positive CDI diagnosis were compared to two control groups: group 1 = diarrhoeic patients negative for C. difficile and matched (1:1) to CDI cases on the ward location and age, and group 2 = diarrhoeic patients colonised with a non-toxigenic strain of C. difficile . Faecal lactoferrin and calprotectin concentrations in faeces were determined for patients with CDI and controls. Of 135 patients with CDI, 87 (64.4%) had a positive stool cytotoxicity assay (free toxin) and 48 (35.6%) had a positive toxigenic culture without detectable toxins in the stools. The median lactoferrin values were 26.8 μg/g, 8.0 μg/g and 15.8 μg/g in CDI patients and groups 1 and 2, respectively. The median calprotectin values were 218.0 μg/g, 111.5 μg/g and 111.3 μg/g, respectively. Among patients with CDI, faecal lactoferrin and calprotectin levels were higher in those with free toxins in their stools (39.2 vs. 10.2 μg/g, p = 0.003 and 274.0 vs. 166.0 μg/g, p = 0.051, respectively). Both faecal calprotectin and lactoferrin were higher in patients with CDI, especially in those with detectable toxin in faeces, suggesting a correlation between intestinal inflammation and toxins in stools.