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.

Intersectoral collaboration in a One Health approach: Lessons learned from a country-level simulation exercise.

Intersectoral collaboration is an essential component of the One Health (OH) approach, which recognises the interconnectedness of the health of humans, animals, and the environment. The OH European Joint Programme (OHEJP) developed a national foodborne outbreak table-top simulation exercise (SimEx) to practice OH capacity and interoperability across the public health, animal health, and food safety sectors, improving OH preparedness for future disease outbreaks. The Portuguese OHEJP SimEx highlighted strengths and weaknesses regarding the roles and functions of available systems, the constraints of existing legislation, the importance of harmonisation and data sharing, and the creation of common main messages adapted to each target sector. However, there is still a long way to go to ensure cooperation among the Public Health, Animal Health, and Food Safety sectors, as a OH approach relies not only on the awareness of "field experts" but also on political and organisational willingness and commitment.

Evaluation of Bile Salts on the Survival and Modulation of Virulence of Aliarcobacter butzleri.

Aliarcobacter butzleri is a Gram-negative bacterium associated with infections of the gastrointestinal tract and widely distributed in various environments. For successful infection, A. butzleri should be able to tolerate various stresses during gastrointestinal passage, such as bile. Bile represents an antimicrobial host barrier that acts against external noxious agents and consists of a variety of bile salts. The intestinal bile salts act as detergents involved in the antimicrobial host defense; although, on the bacterial side, they could also serve as a signal to activate virulence mechanisms. The aim of this work was to understand the effects of bile salts on the survival and virulence of A. butzleri. In our study, A. butzleri was able to survive in the presence of human physiological concentrations of bile salts. Regarding the virulence features, an increase in cellular hydrophobicity, a decrease in motility and expression of flaA gene, as well as an increase in biofilm formation with a concomitant change in the type of biofilm structure were observed in the presence of sub-inhibitory concentration of bile salts. Concerning adhesion and invasion ability, no significant difference was observed. Overall, the results demonstrated that A. butzleri is able to survive in physiological concentrations of bile salts and that exposure to bile salts could change its virulence mechanisms.

Editorial for Special Issue Foodborne Pathogens: Infections and Pathogenesis.

Foodborne microorganisms substantially impact food safety and contribute considerably to the public health and economic burden of infectious diseases worldwide [...].

Recurrent Campylobacter jejuni Infections with In Vivo Selection of Resistance to Macrolides and Carbapenems: Molecular Characterization of Resistance Determinants.

We present two independent cases of recurrent multidrug-resistant Campylobacter jejuni infection in immunocompromised hosts and the clinical challenges encountered due to the development of high-level carbapenem resistance. The mechanisms associated with this unusual resistance for Campylobacters were characterized. Initial macrolide and carbapenem-susceptible strains acquired resistance to erythromycin (MIC > 256mg/L), ertapenem (MIC > 32mg/L), and meropenem (MIC > 32mg/L) during treatment. Carbapenem-resistant isolates developed an in-frame insertion resulting in an extra Asp residue in the major outer membrane protein PorA, within the extracellular loop L3 that connects ß-strands 5 and 6 and forms a constriction zone involved in Ca2+ binding. The isolates presenting the highest MIC to ertapenem exhibited an extra nonsynonymous mutation (G167A|Gly56Asp) at PorA's extracellular loop L1. IMPORTANCE Carbapenem susceptibility patterns suggest drug impermeability, related to either insertion and/or single nucleotide polymorphism (SNP) within porA. Similar molecular events occurring in two independent cases support the association of these mechanisms with carbapenem resistance in Campylobacter spp.

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.

Putative Role of an ABC Efflux System in Aliarcobacter butzleri Resistance and Virulence.

Aliarcobacter butzleri is considered a ubiquitous microorganism and emergent pathogen, for which increasing rates of multidrug resistance have been described. In line with this, the present work aimed to evaluate for the first time the contribution of an ABC efflux system, the YbhFSR, in the resistance and virulence of this bacterium. Following the in silico characterization of the YbhFSR transporter, a mutant strain was constructed by inactivating the gene responsible for ATP-binding. After ensuring that the mutation did not have an impact on bacterial growth, the resistance profile of parental and mutant strains to different antimicrobial agents was evaluated. The results suggest that the efflux pump may influence the resistance to benzalkonium chloride, ethidium bromide, and cadmium, and several other compounds were identified as potential substrates. Regarding the evaluation of the accumulation of ethidium bromide, a slight increase was observed for the mutant strain, demonstrating a potential role of the YbhFSR efflux pump in the extrusion of toxic compounds from A. butzleri. Subsequently, the role of this efflux pump on the A. butzleri known virulence properties was evaluated, but no difference was seen among mutant and parental strains for the motility, biofilm formation ability, susceptibility to oxidative stress, or the ability to adhere and invade Caco-2 cells. However, in contrast to the parental strain, the mutant strain showed a resistance to human serum. Overall, the results support the role of efflux pumps in A. butzleri resistance to antimicrobials, highlighting the particular role of the YbhFSR system.

Effect of Atmospheric Conditions on Pathogenic Phenotypes of Arcobacter butzleri.

Arcobacter butzleri is an emergent gram-negative enteropathogenic bacterium widespread in different environments and hosts. During the colonization of the gastrointestinal tract, bacteria face a variety of environmental conditions to successfully establish infection in a new host. One of these challenges is the fluctuation of oxygen concentrations encountered not only throughout the host gastrointestinal tract and defences but also in the food industry. Oxygen fluctuations can lead to modulations in the virulence of the bacterium and possibly increase its pathogenic potential. In this sense, eight human isolates of A. butzleri were studied to evaluate the effects of microaerobic and aerobic atmospheric conditions in stressful host conditions, such as oxidative stress, acid survival, and human serum survival. In addition, the effects on the modulation of virulence traits, such as haemolytic activity, bacterial motility, biofilm formation ability, and adhesion and invasion of the Caco-2 cell line, were also investigated. Overall, aerobic conditions negatively affected the susceptibility to oxygen reactive species and biofilm formation ability but improved the isolates' haemolytic ability and motility while other traits showed an isolate-dependent response. In summary, this work demonstrates for the first time that oxygen levels can modulate the potential pathogenicity of A. butzleri, although the response to stressful conditions was very heterogeneous among different strains.

Pathogenic Escherichia coli, Salmonella spp. and Campylobacter spp. in Two Natural Conservation Centers of Wildlife in Portugal: Genotypic and Phenotypic Characterization.

Human-wildlife coexistence may increase the potential risk of direct transmission of emergent or re-emergent zoonotic pathogens to humans. Intending to assess the occurrence of three important foodborne pathogens in wild animals of two wildlife conservation centers in Portugal, we investigated 132 fecal samples for the presence of Escherichia coli (Shiga toxin-producing E. coli (STEC) and non-STEC), Salmonella spp. and Campylobacter spp. A genotypic search for genes having virulence and antimicrobial resistance (AMR) was performed by means of PCR and Whole-Genome Sequencing (WGS) and phenotypic (serotyping and AMR profiles) characterization. Overall, 62 samples tested positive for at least one of these species: 27.3% for STEC, 11.4% for non-STEC, 3.0% for Salmonella spp. and 6.8% for Campylobacter spp. AMR was detected in four E. coli isolates and the only Campylobacter coli isolated in this study. WGS analysis revealed that 57.7% (30/52) of pathogenic E. coli integrated genetic clusters of highly closely related isolates (often involving different animal species), supporting the circulation and transmission of different pathogenic E. coli strains in the studied areas. These results support the idea that the health of humans, animals and ecosystems are interconnected, reinforcing the importance of a One Health approach to better monitor and control public health threats.

Repeated out-of-Africa expansions of Helicobacter pylori driven by replacement of deleterious mutations.

Helicobacter pylori lives in the human stomach and has a population structure resembling that of its host. However, H. pylori from Europe and the Middle East trace substantially more ancestry from modern African populations than the humans that carry them. Here, we use a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations, implying selection to remove them during admixture. Simulations show that population fitness can be restored after bottlenecks by migration and subsequent admixture of small numbers of bacteria from non-bottlenecked populations. We conclude that recent spread of African DNA has been driven by deleterious mutations accumulated during the original out-of-Africa bottleneck.

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.

Assessment of the Transmission Dynamics of Clostridioides difficile in a Farm Environment Reveals the Presence of a New Toxigenic Strain Connected to Swine Production.

The recent increase in community-acquired Clostridioides difficile infections discloses the shift in this bacterium epidemiology. This study aimed at establishing a transmission network involving One Health components, as well as assessing the zoonotic potential and genomic features of dominant clones. Samples were collected from different compartments of animal, human and environmental origin, from an animal production unit. C. difficile isolates were characterized for toxigenic profile by multiplex-PCR, while genetic diversity was evaluated by PCR-ribotyping and whole genome-based analysis. The overall C. difficile prevalence was 37.2% (70/188), and included samples from environmental (58.3%, 35/60) and animal (31.5%, 35/111) compartments; human samples (n = 17) taken from healthy workers were negative. A predominant clone from RT033 was found in almost 90% of the positive samples, including samples from all compartments connected to the pig production unit, with core-genome single nucleotide variant (SNV)-based Analysis supporting a clonal transmission between them (mean distance of 0.1 ± 0.1 core-SNVs). The isolates from this clone (herein designated PT RT033) were positive for all C. difficile toxin genes (tcdA, tcdB, cdtA/cdtB). The phyloGenetic positioning of this clone was clearly distinct from the classical RT033 cluster, suggesting a different evolutionary route. This new clone shares genomic features with several RTs from the clade 5 Sequence Type (ST) 11, including a complete pathogenicity locus (PaLoc) that is more similar to the one found in toxigenic strains and contrasting to the less virulent classical RT033 (tcdA-, tcdB-, cdtA + /cdtB +). The presence of a tcdA gene truncated into two ORFs, not previously described, requires further evaluation concerning toxin functionality. We hypothesize that the unique combination of genetic elements found in the PT RT033 clone may contribute to host tropism and environmental dissemination and maintenance. This study constitutes the first report of a toxigenic RT033 clone and adds to the overall knowledge on Clade 5 sequence type 11, considered the C. difficile evolutionary lineage with the highest zoonotic potential. The presence of this clone in all compartments associated with the pig production unit suggests a transmission chain involving these animals and contributes to unveil the role played by animal and environmental reservoirs in this pathogen epidemiology.

Epidemiology of Clostridioides difficile Infection in Portugal: A Retrospective, Observational Study of Hospitalized Patients.

INTRODUCTION: Clostridioides difficile is the main cause of healthcare-associated diarrhea in Europe and North America. The aim of this study was to characterize the epidemiology and clinical burden of Clostridioides difficile infection among hospitalized patients in Portugal. MATERIAL AND METHODS: Retrospective study conducted in six public hospital centers in Portugal. All primary Clostridioides difficile infection episodes and related recurrences occurring in 2017, as well as episodes developing two to eight weeks after the last episode diagnosed in that year, were documented. The National Reference Laboratory (National Institute of Health Dr. Ricardo Jorge) provided national surveillance data on Clostridioides difficile infection. RESULTS: A total of 385 inpatients with at least one primary episode diagnosed in 2017 were included. Most patients were aged over 70 years-old (73.2%). The included patients developed 451 episodes during the observation period. Approximately 44% of primary episodes were community-associated. Most episodes (94.9%) occurred in patients with one or more risk factors, with recent antibiotic exposure being particularly common (86.0%). All-cause in-hospital mortality was 19.5%, being significantly higher in patients aged over 65 years-old versus those aged 18 to 64 years-old (22.4% vs 7.8%, respectively). Over 50 different ribotypes were observed among 206 Clostridioides difficile strains received by the National Reference Laboratory. CONCLUSION: In Portugal, hospitalized patients with Clostridioides difficile infection are mostly older patients presenting risk factors for the development of this infection, particularly recent antibiotic exposure. Mortality is disproportionately high among the older population. Community-associated Clostridioides difficile infection is common among inpatients with this infection.

Introdução: Clostridioides difficile é a principal causa de diarreia nosocomial na Europa e América do Norte. Este estudo teve como objetivo caracterizar a epidemiologia e o impacto clínico da infeção por Clostridioides difficile em doentes hospitalizados em Portugal. Material e Métodos: Estudo retrospetivo conduzido em seis centros hospitalares públicos de Portugal. Foram documentados todos os episódios primários de infeção por Clostridioides difficile ocorridos em 2017 e consequentes recorrências, bem como os episódios que ocorreram entre duas a oito semanas após o último episódio diagnosticado neste ano. Os dados de vigilância nacional de infeção por Clostridioides difficile foram fornecidos pelo laboratório nacional de referência (Instituto Nacional de Saúde Doutor Ricardo Jorge). Resultados: Foram incluídos 385 doentes hospitalizados com pelo menos um episódio primário diagnosticado em 2017. A maioria dos doentes tinha idade igual ou superior a 70 anos (73,2%). Os doentes incluídos tiveram 451 episódios durante o período de observação. Aproximadamente 44% dos episódios primários eram episódios de infeção por Clostridioides difficile adquirida na comunidade. A maioria dos episódios (91,8%) ocorreu em doentes com um ou mais fatores de risco, sendo a exposição recente a antibióticos particularmente comum (86,0%). A mortalidade hospitalar por todas as causas foi de 19,5%, sendo significativamente superior em doentes com idade igual ou superior a 65 anos comparativamente a doentes com idade entre 18 e 64 anos (22,4% versus 7,8%, respetivamente). Mais de 50 ribotipos diferentes foram detetados entre as 206 estirpes de Clostridioides difficile recebidas pelo laboratório nacional de referência. Conclusão: Em Portugal, doentes hospitalizados com infeção por Clostridioides difficile são, na sua maioria, doentes idosos com fatores de risco para o seu desenvolvimento, particularmente exposição recente a antibióticos. A mortalidade é desproporcionalmente elevada na população idosa. Episódios associados à comunidade são comuns em doentes hospitalizados com esta infeção.

Cryptic Prophages Contribution for Campylobacter jejuni and Campylobacter coli Introgression.

Campylobacter coli and C. jejuni, the causing agents of campylobacteriosis, are described to be undergoing introgression events, i.e., the transference of genetic material between different species, with some isolates sharing almost a quarter of its genome. The participation of phages in introgression events and consequent impact on host ecology and evolution remain elusive. Three distinct prophages, named C. jejuni integrated elements 1, 2, and 4 (CJIE1, CJIE2, and CJIE4), are described in C. jejuni. Here, we identified two unreported prophages, Campylobacter coli integrated elements 1 and 2 (CCIE1 and CCIE2 prophages), which are C. coli homologues of CJIE1 and CJIE2, respectively. No induction was achieved for both prophages. Conversely, induction assays on CJIE1 and CJIE2 point towards the inducibility of these prophages. CCIE2-, CJIE1-, and CJIE4-like prophages were identified in a Campylobacter spp. population of 840 genomes, and phylogenetic analysis revealed clustering in three major groups: CJIE1-CCIE1, CJIE2-CCIE2, and CJIE4, clearly segregating prophages from C. jejuni and C. coli, but not from human- and nonhuman-derived isolates, corroborating the flowing between animals and humans in the agricultural context. Punctual bacteriophage host-jumps were observed in the context of C. jejuni and C. coli, and although random chance cannot be fully discarded, these observations seem to implicate prophages in evolutionary introgression events that are modulating the hybridization of C. jejuni and C. coli species.

The Prevalence of Arcobacteraceae in Aquatic Environments: A Systematic Review and Meta-Analysis.

Members of the family Arcobacteraceae are distributed widely in aquatic environments, and some of its species have been associated with human and animal illness. However, information about the diversity and distribution of Arcobacteraceae in different water bodies is still limited. In order to better characterize the health risk posed by members in the family Arcobacteraceae, a systematic review and meta-analysis-based method was used to investigate the prevalence of Arcobacteraceae species in aquatic environments based on available data published worldwide. The database search was performed using related keywords and considering studies up to February 2021. The pooled prevalence in aquatic environments was 69.2%, ranging from 0.6 to 99.9%. These bacteria have a wide geographical distribution, being found in diverse aquatic environments with the highest prevalence found in raw sewage and wastewater treatment plants (WWTP), followed by seawater, surface water, ground water, processing water from food processing plants and water for human consumption. Assessing the effectiveness of treatments in WWTP in eliminating this contamination, it was found that the wastewater treatment may not be efficient in the removal of Arcobacteraceae. Among the analyzed Arcobacteraceae species, Al. butzleri was the most frequently found species. These results highlight the high prevalence and distribution of Arcobacteraceae in different aquatic environments, suggesting a risk to human health. Further, it exposes the importance of identifying and managing the sources of contamination and taking preventive actions to reduce the burden of members of the Arcobacteraceae family.

Genotypic and Phenotypic Characterization of Pathogenic Escherichia coli, Salmonella spp., and Campylobacter spp., in Free-Living Birds in Mainland Portugal.

Birds are potential carriers of pathogens affecting humans and agriculture. Aiming to evaluate the occurrence of the top three most important foodborne pathogens in free-living birds in Portugal, we investigated 108 individual fecal samples from free-living birds and one pooled sample of gull feces (n = 50) for the presence of Escherichia coli (pathogenic and non-pathogenic), Salmonella spp. and Campylobacter spp. Virulence- and antimicrobial resistance- (AMR) associated genes were detected by PCR and Whole-Genome Sequencing (WGS), and phenotypic (serotyping and AMR profiles) characterization was performed. Overall, 8.9% of samples tested positive for pathogenic E. coli, 2.8% for Salmonella spp., and 9.9% for Campylobacter spp. AMR was performed on all pathogenic isolates and in a fraction of non-pathogenic E. coli, being detected in 25.9% of them. Ten of the tested E. coli isolates were multidrug-resistant (MDR), and seven of them were Extended-spectrum ß-lactamase (ESBL) producers. Among Salmonella (n = 3) and Campylobacter (n = 9), only one strain of C. jejuni was identified as MDR. Most of the identified serotypes/sequence types had already been found to be associated with human disease. These results show that free-living birds in Portugal may act as carriers of foodborne pathogens linked to human disease, some of them resistant to critically important antimicrobials.

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).