Insights into macrolide resistance in Arcobacter butzleri: potential resistance mechanisms and impact on bacterial fitness and virulence.

BACKGROUND: Macrolides are recommended for treating the emerging enteropathogen Arcobacter butzleri; nonetheless, this bacterium often exhibits highly variable resistance rates, and the mechanisms behind this resistance phenotype remain largely unexplored. OBJECTIVES: To understand the phenotypic and genotypic consequences associated with the acquisition of erythromycin resistance in A. butzleri, as well as the effects on the fitness of this species. METHODS: Resistant strains resulting from spontaneous mutations and adaptive laboratory evolution under increasing erythromycin concentrations were examined regarding their cross-resistance and collateral susceptibility profiles. Genetic causes of phenotypic antibiotic resistance were analysed by sequencing and bioinformatics, with functional correlation through ethidium bromide accumulation assays. Growth profiles in the presence and absence of erythromycin, motility and biofilm formation abilities were assessed to detect potential changes in fitness and virulence. RESULTS: Clones from spontaneous mutation rate evolution demonstrated decreased susceptibility to erythromycin and other classes of antibiotics, associated with mutations in the transcriptional repressor areR, causing overexpression of the AreABC efflux pump. In turn, WGS analysis of the evolved strain showed additional mutations in the ribosomal proteins L4 and L22 and in the areR gene. Furthermore, the acquisition of macrolide resistance altered A. butzleri virulence and entailed a high biological cost. CONCLUSIONS: The findings of this study have proved that efflux activity contributes synergistically with mutations in the ribosomal proteins L4 and L22 to A. butzleri's high-level macrolide resistance. The results further suggest an impact on the bacterial physiology and virulence, with the increased fitness cost justifying the low worldwide prevalence of high-level resistant circulating strains.

Time trend prevalence of helicobacter pylori infection and endoscopic findings in symptomatic children in Portugal: A retrospective study based on three time points in 2009, 2014, and 2019.

BACKGROUND: Helicobacter pylori infection acquisition occurs mainly in childhood and may be a critical factor in developing long-term complications. In contrast to other developed countries, previous studies have reported a relatively high H. pylori infection prevalence in Portugal, both in children and adults. However, there are no recent data concerning pediatric population. MATERIALS AND METHODS: We performed a retrospective observational study concerning an 11 years period (2009, 2014, 2019), that included patients under 18 years old who underwent upper endoscopy at a pediatric tertiary center. Demographic, clinical-pathological, and microbiological data were collected. RESULTS: Four hundred and sixty one children were included. The average age was 11.7 ± 4.4 years. In total, H. pylori infection was confirmed in 37.3% of cases (histology and/or culture) and a decreasing infection trend was observed (p = .027). The most common indication for endoscopy was abdominal pain, which was a good predictor of infection. Antral nodularity was present in 72.2% of the infected children (p < .001). In the oldest age groups, moderate/severe chronic inflammation, H. pylori density and lymphoid aggregates/follicles were positive predictors for the presence of antral nodularity. For all ages, the presence of antral nodularity, neutrophilic activity in the antrum and corpus and lymphoid follicles/aggregates in the antrum were positive predictors for the presence of H. pylori infection. Among the 139 strains tested for antibiotic susceptibility, 48.9% were susceptible to all tested antibiotics. Resistance to clarithromycin, metronidazole, and both was detected in 23.0%, 12.9%, and 6.5% of the strains, respectively; furthermore, resistance to ciprofloxacin and to amoxicillin was observed in 5.0% and 1.4% of the strains, respectively. CONCLUSIONS: The present study reports (for the first time in Portugal) a significant decreasing trend in the prevalence of pediatric H. pylori infection, although it remains relatively high compared to the recently reported prevalence in other South European countries. We confirmed a previously recognized positive association of some endoscopic and histological features with H. pylori infection, as well as a high prevalence rate of resistance to clarithromycin and to metronidazole. The clinical relevance of these findings requires confirmation with further studies at a national level, taking into account the high incidence rate of gastric cancer in Portugal and the potential need for country-specific intervention strategies.

Helicobacter pylori infection: from standard to alternative treatment strategies.

Helicobacter pylori is the major component of the gastric microbiome of infected individuals and one of the aetiological factors of chronic gastritis, peptic ulcer disease and gastric cancer. The increasing resistance to antibiotics worldwide has made the treatment of H. pylori infection a challenge. As a way to overhaul the efficacy of currently used H. pylori antibiotic-based eradication therapies, alternative treatment strategies are being devised. These include probiotics and prebiotics as adjuvants in H. pylori treatment, antimicrobial peptides as alternatives to antibiotics, photodynamic therapy ingestible devices, microparticles and nanoparticles applied as drug delivery systems, vaccines, natural products, and phage therapy. This review provides an updated synopsis of these emerging H. pylori control strategies and discusses the advantages, hurdles, and challenges associated with their development and implementation. An effective human vaccine would be a major achievement although, until now, projects regarding vaccine development have failed or were discontinued. Numerous natural products have demonstrated anti-H. pylori activity, mostly in vitro, but further clinical studies are needed to fully disclose their role in H. pylori eradication. Finally, phage therapy has the potential to emerge as a valid alternative, but major challenges remain, namely the isolation of more H. pylori strictly virulent bacterio(phages).

Airborne spores' dissemination of a swine associated Clostridioides difficile clone.

This study supports the airborne dissemination of Clostridioides difficile spores. Of the sieve impaction samples collected at a swine production unit, 66.7% were positive and all belonged to the predominantly established clone. Spores' density varied according to the characteristics of the animal population, suggesting the possibility of airborne transmission.

Characterization and Genomic Analysis of a New Phage Infecting Helicobacter pylori.

Helicobacter pylori, a significant human gastric pathogen, has been demonstrating increased antibiotic resistance, causing difficulties in infection treatment. It is therefore important to develop alternatives or complementary approaches to antibiotics to tackle H. pylori infections, and (bacterio)phages have proven to be effective antibacterial agents. In this work, prophage isolation was attempted using H. pylori strains and UV radiation. One phage was isolated and further characterized to assess potential phage-inspired therapeutic alternatives to H. pylori infections. HPy1R is a new podovirus prophage with a genome length of 31,162 bp, 37.1% GC, encoding 36 predicted proteins, of which 17 were identified as structural. Phage particles remained stable at 37 °C, from pH 3 to 11, for 24 h in standard assays. Moreover, when submitted to an in vitro gastric digestion model, only a small decrease was observed in the gastric phase, suggesting that it is adapted to the gastric tract environment. Together with its other characteristics, its capability to suppress H. pylori population levels for up to 24 h post-infection at multiplicities of infection of 0.01, 0.1, and 1 suggests that this newly isolated phage is a potential candidate for phage therapy in the absence of strictly lytic phages.

Characterization of AreABC, an RND-Type Efflux System Involved in Antimicrobial Resistance of Aliarcobacter butzleri.

Aliarcobacter butzleri is an emergent enteropathogen for which resistance to several classes of antimicrobial agents has been described, although the underlying mechanisms have been poorly addressed. We aimed to evaluate the contribution of the resistance-nodulation-division-type (RND) efflux system, AreABC, to drug resistance in A. butzleri. A. butzleri strains were first tested against several antimicrobials with and without an efflux pump inhibitor. Then, erythromycin-resistant strains were screened for the presence of a premature stop codon in a putative transcriptional regulator of the AreABC system, areR. Lastly, antimicrobial susceptibility and ethidium bromide (EtBr) accumulation were evaluated using an areB knockout strain and a strain overexpressing the AreABC system through areR truncation. The presence of the efflux pump inhibitor resulted in increased susceptibility to most of the antimicrobials tested. A correlation between erythromycin resistance and the presence of premature stop codons in areR was observed. The truncation of areR resulted in increased expression of the AreABC system and decreased susceptibility to various antimicrobials. In contrast, areB inactivation resulted in increased susceptibility and a higher intracellular accumulation of EtBr. In conclusion, the AreABC efflux pump plays a role in the resistance of A. butzleri to multiple drugs and is regulated by a putative transcriptional repressor, areR. Our results support the importance of efflux pumps in this bacterium's resistance to major classes of antibiotics and other antimicrobials.

Helicobacter pylori Infection in Pediatric Patients Living in Europe: Results of the EuroPedHP Registry 2013 to 2016.

OBJECTIVES: The aim of the study was to assess clinical presentation, endoscopic findings, antibiotic susceptibility and treatment success of Helicobacter pylori (H. pylori) infected pediatric patients. METHODS: Between 2013 and 2016, 23 pediatric hospitals from 17 countries prospectively submitted data on consecutive H. pylori-infected (culture positive) patients to the EuroPedHP-Registry. RESULTS: Of 1333 patients recruited (55.1% girls, median age 12.6 years), 1168 (87.6%) were therapy naïve (group A) and 165 (12.4%) had failed treatment (group B). Patients resided in North/Western (29.6%), Southern (34.1%) and Eastern Europe (23.0%), or Israel/Turkey (13.4%). Main indications for endoscopy were abdominal pain or dyspepsia (81.2%, 1078/1328). Antral nodularity was reported in 77.8% (1031/1326) of patients, gastric or duodenal ulcers and erosions in 5.1% and 12.8%, respectively. Primary resistance to clarithromycin (CLA) and metronidazole (MET) occurred in 25% and 21%, respectively, and increased after failed therapy. Bacterial strains were fully susceptible in 60.5% of group A, but in only 27.4% of group B. Primary CLA resistance was higher in Southern and Eastern Europe (adjusted odds ratio [ORadj] = 3.44, 95% confidence interval [CI] 2.22-5.32, P < 0.001 and 2.62, 95% CI: 1.63-4.22, P < 0.001, respectively) compared with Northern/Western Europe. Children born outside Europe showed higher primary MET resistance (ORadj = 3.81, 95% CI: 2.25-6.45, P < 0.001). Treatment success in group A reached only 79.8% (568/712) with 7 to 14 days triple therapy tailored to antibiotic susceptibility. CONCLUSIONS: Peptic ulcers are rare in dyspeptic H. pylori-infected children. Primary resistance to CLA and MET is markedly dependent on geographical regions of birth and residence. The ongoing survey will show whether implementation of the updated ESPGHAN/NASPGHAN guidelines will improve the eradication success.

Imipenem Resistance in Clostridium difficile Ribotype 017, Portugal.

We describe imipenem-resistant and imipenem-susceptible clinical isolates of Clostridium difficile ribotype 017 in Portugal. All ribotype 017 isolates carried an extra penicillin-binding protein gene, pbp5, and the imipenem-resistant isolates had additional substitutions near the transpeptidase active sites of pbp1 and pbp3. These clones could disseminate and contribute to imipenem resistance.

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants.

A whole-genome sequencing (WGS) approach was conducted in order to identify the molecular determinants associated with antimicrobial resistance in 12 multidrug-resistant Campylobacter jejuni and Campylobacter coli isolates, with a focus on aminoglycoside resistance determinants. Two variants of a new aminoglycoside phosphotransferase gene [aph(2″)-Ii1 and aph(2″)-Ii2 ] putatively associated with gentamicin resistance were found. In addition, the following new genes were identified for the first time in Campylobacter: a lincosamide nucleotidyltransferase gene [lnu(G)], likely associated with lincomycin resistance, and two resistance enzyme genes (spw and apmA) similar to those found in Staphylococcus aureus, which may confer spectinomycin and gentamicin resistance, respectively. A C1192T mutation of the 16S rRNA gene that may be involved in spectinomycin resistance was also found in a C. coli isolate. Genes identified in the present study were located either on the bacterial chromosome or on plasmids that could be transferred naturally. Their role in aminoglycoside resistance remains to be supported by genetic studies. Regarding the other antimicrobial agents studied, i.e., ampicillin, ciprofloxacin, erythromycin, and tetracycline, a perfect correlation between antimicrobial phenotypes and genotypes was found. Overall, our data suggest that WGS analysis is a powerful tool for identifying resistance determinants in Campylobacter and can disclose the full genetic elements associated with resistance, including antimicrobial compounds not tested routinely in antimicrobial susceptibility testing.

Acute gastroenteritis outbreak associated to norovirus GI.9 in a Portuguese army base.

Gastroenteritis is considered a major illness within the military settings being caused by foodborne enteric pathogens that are particularly easily spread in the crowded conditions of military camps. Gastroenteritis outbreaks caused by norovirus usually affect a great number of soldiers due to the low infectious dose, copious viral shedding, and environmental stability. The present study describes the investigation of an outbreak of acute gastroenteritis that occurred in April 2015 in a Portuguese army base, focusing on the study of the epidemiological curve, symptoms experienced by the affected soldiers, and results of food, water, and stool microbiological analysis. From a total of 938 military personnel stationed on the base 46 soldiers developed acute gastroenteritis. Stool analysis of seven cases showed to be positive for norovirus GI.9 that was the probable cause of the outbreak. This report shows that genogroup I norovirus can also cause considerable morbidity in healthy young soldiers, affecting the operational effectiveness on the military forces. J. Med. Virol. 89:922-925, 2017. © 2016 Wiley Periodicals, Inc.

Population genetic structure of Helicobacter pylori strains from Portuguese-speaking countries.

BACKGROUND: The human gastric colonizer Helicobacter pylori is useful to track human migrations given the agreement between the bacterium phylogeographic distribution and human migrations. As Portugal was an African and Brazilian colonizer for over 400 years, we hypothesized that Portuguese isolates were likely genetically closer with those from countries colonized by Portuguese in the past. We aimed to characterize the population structure of several Portuguese-speaking countries, including Portugal, Brazil, Angola, and Cape Verde. MATERIALS AND METHODS: We included strains isolated in Portugal from Portuguese and from former Portuguese colonies. These strains were typed by multilocus sequence typing (MLST) for seven housekeeping genes. We also retrieved from Multi Locus Sequence Typing Web site additional housekeeping gene sequences, namely from Angola and Brazil. RESULTS: We provided evidence that strains from Portuguese belong to hpEurope and that the introgression of hpEurope in non-European countries that speak Portuguese is low, except for Brazil and Cape Verde, where hpEurope accounted for one quarter and one half of the population, respectively. We found genetic similarity for all strains from Portuguese-speaking countries that belong to hpEurope population. Moreover, these strains showed a predominance of ancestral Europe 2 (AE2) over ancestral Europe 1 (AE1), followed by ancestral Africa 1. CONCLUSIONS: H. pylori is a useful marker even for relative recent human migration events and may become rapidly differentiated from founder populations. H. pylori from Portuguese-speaking countries assigned to hpEurope appears to be a hybrid population resulting from the admixture of AE1, AE2 and ancestral hpAfrica1.

The expression of Helicobacter pylori tfs plasticity zone cluster is regulated by pH and adherence, and its composition is associated with differential gastric IL-8 secretion.

BACKGROUND: Helicobacter pylori virulence is associated with different clinical outcomes. The existence of an intact dupA gene from tfs4b cluster has been suggested as a predictor for duodenal ulcer development. However, the role of tfs plasticity zone clusters in the development of ulcers remains unclear. We studied several H. pylori strains to characterize the gene arrangement of tfs3 and tfs4 clusters and their impact in the inflammatory response by infected gastric cells. METHODS: The genome of 14 H. pylori strains isolated from Western patients, pediatric (n=10) and adult (n=4), was fully sequenced using the Illumina platform MiSeq, in addition to eight pediatric strains previously sequenced. These strains were used to infect human gastric cells, and the secreted interleukin-8 (IL-8) was quantified by ELISA. The expression of virB2, dupA, virB8, virB10, and virB6 was assessed by quantitative PCR in adherent and nonadherent fractions of H. pylori during in vitro co-infection, at different pH values. RESULTS: We have found that cagA-positive H. pylori strains harboring a complete tfs plasticity zone cluster significantly induce increased production of IL-8 from gastric cells. We have also found that the region spanning from virB2 to virB10 genes constitutes an operon, whose expression is increased in the adherent fraction of bacteria during infection, as well as in both adherent and nonadherent fractions at acidic conditions. CONCLUSIONS: A complete tfs plasticity zone cluster is a virulence factor that may be important for the colonization of H. pylori and to the development of severe outcomes of the infection with cagA-positive strains.

Insights in the pathogenesis and resistance of Arcobacter: A review.

Arcobacter genus currently comprises 18 recognized species, among which Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii have been associated with human and animal disease. Although these organisms, with special emphasis A. butzleri, are emerging as clinical pathogens, several aspects of their epidemiology and virulence are only starting to be clarified. In vitro human and animal cell culture assays have been used to show that several Arcobacter species can adhere to and invade eukaryotic cells, induce an immune response and produce toxins that damage host cells. In addition, data from genome sequencing highlighted several potential markers that may be helpful candidates for the study and understanding of these mechanisms; however, more work is necessary to clarify the molecular mechanisms involved in Arcobacter virulence. Arcobacter can be considered a relatively robust organism showing to be able to survive in adverse conditions, as the ones imposed by food processing and storage. Moreover, these bacteria have shown increased antibiotic resistance, along with high multidrug resistance. In this review, we seek to update the state-of-the-art concerning Arcobacter distribution, its interaction with the host, the trends of antibiotic resistance, its ability to survive, and finally the use of natural antimicrobials for control of Arcobacter.

Helicobacter pullorum isolated from fresh chicken meat: antibiotic resistance and genomic traits of an emerging foodborne pathogen.

Meat and meat products are important sources of human intestinal infections. We report the isolation of Helicobacter pullorum strains from chicken meat. Bacteria were isolated from 4 of the 17 analyzed fresh chicken meat samples, using a membrane filter method. MIC determination revealed that the four strains showed acquired resistance to ciprofloxacin; one was also resistant to erythromycin, and another one was resistant to tetracycline. Whole-genome sequencing of the four strains and comparative genomics revealed important genetic traits within the H. pullorum species, such as 18 highly polymorphic genes (including a putative new cytotoxin gene), plasmids, prophages, and a complete type VI secretion system (T6SS). The T6SS was found in three out of the four isolates, suggesting that it may play a role in H. pullorum pathogenicity and diversity. This study suggests that the emerging pathogen H. pullorum can be transmitted to humans by chicken meat consumption/contact and constitutes an important contribution toward a better knowledge of the genetic diversity within the H. pullorum species. In addition, some genetic traits found in the four strains provide relevant clues to how this species may promote adaptation and virulence.

Helicobacter pullorum cytolethal distending toxin targets vinculin and cortactin and triggers formation of lamellipodia in intestinal epithelial cells.

Helicobacter pullorum, a bacterium initially isolated from poultry, has been associated with human digestive disorders. However, the factor responsible for its cytopathogenic effects on epithelial cells has not been formally identified. The cytopathogenic alterations induced by several human and avian H. pullorum strains were investigated on human intestinal epithelial cell lines. Moreover, the effects of the cytolethal distending toxin (CDT) were evaluated first by using a wild-type strain and its corresponding cdtB isogenic mutant and second by delivering the active CdtB subunit of the CDT directly into the cells. All of the H. pullorum strains induced cellular distending phenotype, actin cytoskeleton remodeling, and G2/M cell cycle arrest. These effects were dependent on the CDT, as they were (1) not observed in response to a cdtB isogenic mutant strain and (2) present in cells expressing CdtB. CdtB also induced an atypical delocalization of vinculin from focal adhesions to the perinuclear region, formation of cortical actin-rich large lamellipodia with an upregulation of cortactin, and decreased cellular adherence. In conclusion, the CDT of H. pullorum is responsible for major cytopathogenic effects in vitro, confirming its role as a main virulence factor of this emerging human pathogen.

Genotypic and phenotypic features of Arcobacter butzleri pathogenicity.

Even though Arcobacter butzleri has been implicated in some human disease as diarrhoea and bacteraemia, much of its pathogenesis and virulence factors remain unclear. In this work we have compared pathogenic and genotypic properties of six A. butzleri isolates from human and non-human sources. The tested isolates showed to be susceptible to tetracyclines and aminoglycosides, however non-human isolates were all resistant to quinolones. The ability to form biofilms was variable among the tested strains, and all of them showed a weak haemolytic activity. The presence of nine putative virulence genes was determined, with cadF, ciaB, cj1349, mviN, pldA, tlyA being detected in all strains, while irgA (3/6), hecA (5/6), hecB (4/6) were detected only in some strains. High levels of adhesion were observed for A. butzleri on Caco-2 cells, with pre-existing inflammation showing no significant effect on the adherence ability; yet variable levels of invasion were observed. A. butzleri isolates were able to survive intracellularly in Caco-2 cells and to induce a significant up-regulation of interleukin-8 secretion and structural cell rearrangements. These data brings new insights on A. butzleri virulence and highlights its pathogenic potential.

Outbreak of Clostridium difficile PCR ribotype 027--the recent experience of a regional hospital.

BACKGROUND: Clostridium difficile infection (CDI) is the leading cause of healthcare-associated diarrhea, and several outbreaks with increased severity and mortality have been reported. In this study we report a C. difficile PCR ribotype 027 outbreak in Portugal, aiming to contribute to a better knowledge of the epidemiology of this agent in Europe. METHODS: Outbreak report with retrospective study of medical records and active surveillance data of all inpatients with the diagnosis of CDI, from 1st January to 31th December 2012, in a Portuguese hospital. C. difficile isolates were characterized regarding ribotype, toxin genes and moxifloxin resistance. Outbreak control measures were taken, concerning communication, education, reinforcement of infection control measures, optimization of diagnosis and treatment of CDI, and antibiotic stewardship. RESULTS: Fifty-three inpatients met the case definition of C. difficile-associated infection: 55% males, median age was 78.0 years (interquartile range: 71.0-86.0), 75% had co-morbidities, only 15% had a nonfatal condition, 68% had at least one criteria of severe disease at diagnosis, 89% received prior antibiotherapy, 79% of episodes were nosocomial. CDI rate peak was 13.89/10,000 bed days. Crude mortality rate at 6 months was 64.2% while CDI attributable cause was 11.3%. Worse outcome was related to older age (P = 0.022), severity criteria at diagnosis (leukocytosis (P = 0.008) and renal failure), and presence of fatal underlying condition (P = 0.025). PCR ribotype 027 was identified in 16 of 22 studied samples. CONCLUSIONS: This is the first report of a 027-CDI outbreak in Portugal. We emphasize the relevance of the measures taken to control the outbreak and highlight the importance of implementing a close and active surveillance of CDI.

Epidemiological Data and Antimicrobial Resistance of Campylobacter spp. in Portugal from 13 Years of Surveillance.

This study extensively analyzed campylobacteriosis surveillance in Portugal from 2009 to 2021, aiming to investigate demographic shifts, seasonal variations, and antimicrobial resistance (AMR) within Campylobacter isolates. Surveillance network and sentinel laboratory-based system data revealed a substantial under-notification of campylobacteriosis cases, suggesting an underestimated disease burden. Notification rates exhibited a paradigm shift, with a notable prevalence among the pediatric population, particularly in children aged 1-4 years, diverging from European reports. Additionally, an emerging trend of Campylobacter infections in younger adults (15-44 years) was observed. The study unveiled a unique seasonal distribution of cases, defying typical summer peaks seen elsewhere. AMR analysis revealed high resistance to ciprofloxacin and tetracycline, in both C. jejuni (93.7% and 79.2%, respectively) and C. coli (96.5% and 93.2%, respectively), stable throughout the studied period (2013-2021). C. coli exhibited significantly higher resistance to erythromycin, gentamicin, ampicillin and ertapenem compared to C. jejuni (p < 0.001). Multilocus Sequence Typing (MLST) data demonstrated the distribution of resistance markers across diverse sequence types, challenging the notion of a clonal origin for multidrug-resistant isolates. In conclusion, the study highlights the need for enhanced surveillance and raises concerns about alarming AMR levels, recommending the implementation of whole-genome sequencing (WGS)-based surveillance for a deeper comprehension of disease patterns and an evolving AMR landscape.

Genomic study of European Clostridioides difficile ribotype 002/sequence type 8.

Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.

Validation of a fluorescence in situ hybridization method using peptide nucleic acid probes for detection of Helicobacter pylori clarithromycin resistance in gastric biopsy specimens.

Here, we evaluated a previously established peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method as a new diagnostic test for Helicobacter pylori clarithromycin resistance detection in paraffin-embedded gastric biopsy specimens. Both a retrospective study and a prospective cohort study were conducted to evaluate the specificity and sensitivity of a PNA-FISH method to determine H. pylori clarithromycin resistance. In the retrospective study (n = 30 patients), full agreement between PNA-FISH and PCR-sequencing was observed. Compared to the reference method (culture followed by Etest), the specificity and sensitivity of PNA-FISH were 90.9% (95% confidence interval [CI], 57.1% to 99.5%) and 84.2% (95% CI, 59.5% to 95.8%), respectively. In the prospective cohort (n = 93 patients), 21 cases were positive by culture. For the patients harboring clarithromycin-resistant H. pylori, the method showed sensitivity of 80.0% (95% CI, 29.9% to 98.9%) and specificity of 93.8% (95% CI, 67.7% to 99.7%). These values likely represent underestimations, as some of the discrepant results corresponded to patients infected by more than one strain. PNA-FISH appears to be a simple, quick, and accurate method for detecting H. pylori clarithromycin resistance in paraffin-embedded biopsy specimens. It is also the only one of the methods assessed here that allows direct and specific visualization of this microorganism within the biopsy specimens, a characteristic that allowed the observation that cells of different H. pylori strains can subsist in very close proximity in the stomach.