Enteroaggregative Escherichia coli as etiological agent of endemic diarrhea in Spain: A prospective multicenter prevalence study with molecular characterization of isolates.

Background: Enteroaggregative Escherichia coli (EAEC) is increasingly associated with domestically acquired diarrheal episodes in high-income countries, particularly among children. However, its specific role in endemic diarrhea in this setting remains under-recognized and information on molecular characteristics of such EAEC strains is limited. We aimed to investigate the occurrence of EAEC in patients with non-travel related diarrhea in Spain and molecularly characterize EAEC strains associated with illness acquired in this high-income setting. Methods: In a prospective multicenter study, stool samples from diarrheal patients with no history of recent travel abroad (n = 1,769) were collected and processed for detection of EAEC and other diarrheagenic E. coli (DEC) pathotypes by PCR. An additional case-control study was conducted among children ≤5 years old. Whole-genome sequences (WGS) of the resulting EAEC isolates were obtained. Results: Detection of DEC in the study population. DEC was detected in 23.2% of patients aged from 0 to 102 years, with EAEC being one of the most prevalent pathotypes (7.8%) and found in significantly more patients ≤5 years old (9.8% vs. 3.4%, p < 0.001). Although not statistically significant, EAEC was more frequent in cases than in controls. WGS-derived characterization of EAEC isolates. Sequence type (ST) 34, ST200, ST40, and ST10 were the predominant STs. O126:H27, O111:H21, and O92:H33 were the predominant serogenotypes. Evidence of a known variant of aggregative adherence fimbriae (AAF) was found in 89.2% of isolates, with AAF/V being the most frequent. Ten percent of isolates were additionally classified as presumptive extraintestinal pathogenic E. coli (ExPEC), uropathogenic E. coli (UPEC), or both, and belonged to clonal lineages that could be specifically associated with extraintestinal infections. Conclusion: EAEC was the only bacterial enteric pathogen detected in a significant proportion of cases of endemic diarrhea in Spain, especially in children ≤5 years old. In particular, O126:H27-ST200, O111:H21-ST40, and O92:H33-ST34 were the most important subtypes, with all of them infecting both patients and asymptomatic individuals. Apart from this role as an enteric pathogen, a subset of these domestically acquired EAEC strains revealed an additional urinary/systemic pathogenic potential.

Executive summary of the consensus statement of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Spanish Society of Neurology (SEN), Spanish Society of Immunology (SEI), Spanish Society of Pediatric Infectology (SEIP), Spanish Society of Rheumatology (SER), and Spanish Academy of Dermatology and Venereology (AEDV), on the diagnosis, treatment and prevention of Lyme borreliosis.

The diagnosis of Lyme borreliosis (LB) is based on the epidemiological history, clinical manifestations and microbiological findings in the early disseminated and late phases of the disease. Related to this fact, microbiological diagnostic techniques have recently appeared. Far from facilitating the diagnosis and the clinical-therapeutic management of LB patients, they are generating confusion. Herein, experts and representatives of Spanish Scientific Societies [Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Spanish Society of Neurology (SEN), Spanish Society of Immunology (SEI), Spanish Society of Pediatric Infectology (SEIP), Spanish Society of Rheumatology (SER), and Spanish Academy of Dermatology and Venereology (AEDV)] exposed the executive summary after reviewing the epidemiology, clinical spectrum, available diagnostic techniques for the diagnosis of Borrelia burgdorferi infection, therapeutic and prevention options of LB. By consensus, recommendations for microbiological diagnosis are offered together with those supporting the therapeutic management and prophylaxis of infection.

Executive summary of the consensus statement of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Spanish Society of Neurology (SEN), Spanish Society of Immunology (SEI), Spanish Society of Pediatric Infectology (SEIP), Spanish Society of Rheumatology (SER), and Spanish Academy of Dermatology and Venereology (AEDV), on the diagnosis, treatment and prevention of Lyme borreliosis / Resumen ejecutivo del documento de consenso de la SEIMC, SEN, SEI, SEIP, SER y AEDV sobre el diagnóstico, tratamiento y prevención de la borreliosis de Lyme

The diagnosis of Lyme borreliosis (LB) is based on the epidemiological history, clinical manifestations and microbiological findings in the early disseminated and late phases of the disease. Related to this fact, microbiological diagnostic techniques have recently appeared. Far from facilitating the diagnosis and the clinical-therapeutic management of LB patients, they are generating confusion. Herein, experts and representatives of Spanish Scientific Societies [Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), Spanish Society of Neurology (SEN), Spanish Society of Immunology (SEI), Spanish Society of Pediatric Infectology (SEIP), Spanish Society of Rheumatology (SER), and Spanish Academy of Dermatology and Venereology (AEDV)] exposed the executive summary after reviewing the epidemiology, clinical spectrum, available diagnostic techniques for the diagnosis of Borrelia burgdorferi infection, therapeutic and prevention options of LB. By consensus, recommendations for microbiological diagnosis are offered together with those supporting the therapeutic management and prophylaxis of infection.(AU)

El diagnóstico de la borreliosis de Lyme (BL) se basa en la historia epidemiológica, las manifestaciones clínicas y los hallazgos microbiológicos de las etapas temprana diseminada y tardía de la enfermedad. En relación a este hecho, han aparecido recientemente técnicas diagnósticas microbiológicas que, lejos de facilitar el diagnóstico y el manejo clínico-terapéutico de los pacientes con BL, están generando confusión. Por ello, los expertos y representantes de las sociedades científicas españolas [Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Sociedad Española de Neurología (SEN), Sociedad Española de Inmunología (SEI), Sociedad Española de Infectología Pediátrica (SEIP), Sociedad Española de Reumatología (SER) y Academia Española de Dermatología y Venereología (AEDV)] han presentado el documento de síntesis tras revisar la epidemiología, el espectro clínico, las técnicas disponibles para el diagnóstico de la infección por Borrelia burgdorferi, así como las opciones terapéuticas y preventivas de BL. De manera consensuada, se ofrecen recomendaciones para el diagnóstico microbiológico, así como recomendaciones que respaldan el manejo terapéutico y la profilaxis de la infección.(AU)

Strengthening the genomic surveillance of Francisella tularensis by using culture-free whole-genome sequencing from biological samples.

Introduction: Francisella tularensis is a highly infectious bacterium that causes the zoonotic disease tularemia. The development of genotyping methods, especially those based on whole-genome sequencing (WGS), has recently increased the knowledge on the epidemiology of this disease. However, due to the difficulties associated with the growth and isolation of this fastidious pathogen in culture, the availability of strains and subsequently WGS data is still limited. Methods: To surpass these constraints, we aimed to implement a culture-free approach to capture and sequence F. tularensis genomes directly from complex samples. Biological samples obtained from 50 common voles and 13 Iberian hares collected in Spain were confirmed as positive for F. tularensis subsp. holarctica and subjected to a WGS target capture and enrichment protocol, using RNA oligonucleotide baits designed to cover F. tularensis genomic diversity. Results: We obtained full genome sequences of F. tularensis from 13 animals (20.6%), two of which had mixed infections with distinct genotypes, and achieved a higher success rate when compared with culture-dependent WGS (only successful for two animals). The new genomes belonged to different clades commonly identified in Europe (B.49, B.51 and B.262) and subclades. Despite being phylogenetically closely related to other genomes from Spain, the detected clusters were often found in other countries. A comprehensive phylogenetic analysis, integrating 599 F. tularensis subsp. holarctica genomes, showed that most (sub)clades are found in both humans and animals and that closely related strains are found in different, and often geographically distant, countries. Discussion: Overall, we show that the implemented culture-free WGS methodology yields timely, complete and high-quality genomic data of F. tularensis, being a highly valuable approach to promote and potentiate the genomic surveillance of F. tularensis and ultimately increase the knowledge on the genomics, ecology and epidemiology of this highly infectious pathogen.

Genetic Traces of the Francisella tularensis Colonization of Spain, 1998-2020.

More than 1000 humans have acquired the febrile disease tularemia in Spain since the first notification of human cases in 1997. We here aimed to study the recent molecular evolution of the causative bacterium Francisella tularensis during disease establishment in Spain. Single-nucleotide polymorphisms (SNPs) and variable-number tandem repeats (VNTRs) were analyzed in whole-genome sequences (WGS) of F. tularensis. Short-read WGS data for 20 F. tularensis strains from humans infected in the periods 2014-2015 and 2018-2020 in Spain were generated. These data were combined with WGS data of 25 Spanish strains from 1998 to 2008 and two reference strains. Capillary electrophoresis data of VNTR genetic regions were generated and compared with the WGS data for the 11 strains from 2014 to 2015. Evolutionary relationships among strains were analyzed by phylogenetic methods. We identified 117 informative SNPs in a 1,577,289-nucleotide WGS alignment of 47 F. tularensis genomes. Forty-five strains from Spain formed a star-like SNP phylogeny with six branches emerging from a basal common node. The most recently evolved genomes formed four additional star-like structures that were derived from four branches of the basal common node. VNTR copy number variation was detected in two out of 10 VNTR regions examined. Genetic clustering of strains by VNTRs agreed with the clustering by SNPs. The SNP data provided higher resolution among strains than the VNTRs data in all but one cases. There was an excellent correlation between VNTR marker sizing by capillary electrophoresis and prediction from WGS data. The genetic data strongly support that tularemia, indeed, emerged recently in Spain. Distinct genetic patterns of local F. tularensis population expansions imply that the pathogen has colonized a previously disease-free geographical area. We also found that genome-wide SNPs provide higher genetic resolution among F. tularensis genomes than the use of VNTRs, and that VNTR copy numbers can be accurately predicted using short-read WGS data.

Phylogeography and Genetic Diversity of Francisella tularensis subsp. holarctica in France (1947-2018).

In France, tularemia is caused by Francisella tularensis subsp. holarctica and is a sporadic disease affecting mainly wildlife animals and humans. F. tularensis species presents low genetic diversity that remains poorly described in France, as only a few genomes of isolates from the country are available so far. The objective of this study was to characterize the genetic diversity of F. tularensis in France and describe the phylogenetic distribution of isolates through whole-genome sequencing and molecular typing. Whole genomes of 350 strains of human or animal origin, collected from 1947 to 2018 in France and neighboring countries, were sequenced. A preliminary classification using the established canonical single nucleotide polymorphism (canSNP) nomenclature was performed. All isolates from France (except four) belonged to clade B.44, previously described in Western Europe. To increase the resolution power, a whole-genome SNP analysis was carried out. We were able to accurately reconstruct the population structure according to the global phylogenetic framework, and highlight numerous novel subclades. Whole-genome SNP analysis identified 87 new canSNPs specific to these subclades, among which 82 belonged to clade B.44. Identifying genomic features that are specific to sublineages is highly relevant in epidemiology and public health. We highlighted a large number of clusters among a single clade (B.44), which shows for the first time some genetic diversity among F. tularensis isolates from France, and the star phylogeny observed in clade B.44-subclades revealed that F. tularensis biodiversity in the country is relatively recent and resulted from clonal expansion of a single population. No association between clades and hosts or clinical forms of the disease was detected, but spatiotemporal clusters were identified for the first time in France. This is consistent with the hypothesis of persistence of F. tularensis strains found in Western Europe in the environment, associated with slow replication rates. Moreover, the presence of identical genotypes across long periods of time, and across long distances, supports this hypothesis but also suggests long-distance dispersal of the bacterium.

Rapid and Highly Sensitive DNA Flow Technology Platform to Detect Tick-Borne Bacterial Pathogens in Clinical Samples.

Zoonotic diseases represent a significant public health concern worldwide due to the emergence/re-emergence of vector-borne diseases in the last decade. Ticks are the most important vectors in the northern hemisphere and can transmit diseases such as Lyme disease, human granulocytic anaplasmosis, and spotted fever rickettsioses, among others. Therefore, there is a growing need to develop better and faster diagnostic tools that can detect zoonotic human pathogens in clinical samples. In this study, we present the results for a new kit tick-borne bacteria flow chip (TBFC), which allows the simultaneous screening of seven different bacterial pathogens in human samples using a DNA flow technology platform (hybriSpot system). The analytical sensitivity and specificity of the TBFC were calculated spiking bacterial DNA in human DNA samples, and the results were compared with an in-house single PCR-reverse line blot (RLB) routinely used for diagnosis at the National Center for Microbiology in Spain. The analytical sensitivity and specificity of the TBFC were almost identical to the PCR-RLBs used in diagnosis. In addition, samples from patients (n = 212) with a wide range of clinical signs/symptoms consistent with multisystem disorders suggestive of a tick-borne infection were tested using the TBFC, and the results were compared with those obtained by PCR-RLB. The concordance of both methods using patient samples was 97.2%. The TBFC kit is a rapid new and cost-efficient diagnostic molecular tool capable of detecting tick-borne pathogens in clinical samples.

An unexpected cause of dysphagia: pleural mesothelioma.

Malignant mesothelioma usually originates from the pleura or peritoneum, and has a poor prognosis. The incidence of this type of tumor is increasing worldwide, which is probably a result of occupational or environmental exposure to asbestos. In 90% dyspnea, chest pain or a combination of both are usually the initial symptoms. Dysphagia only occurs in 1.4% and is very rare as the initial symptom. We present the case of a middle-aged patient, in whom the initial symptom was dysphagia, so an endoscopy was performed. This showed extrinsic compression of the esophagus that was demonstrated when performing the chest X-ray, in which it was revealed a posterior mediastinal mass surrounding the esophagus concentrically without mucosal invasion.

Zoonotic Bacteria in Fleas Parasitizing Common Voles, Northwestern Spain.

We detected Francisella tularensis and Bartonella spp. in fleas parasitizing common voles (Microtus arvalis) from northwestern Spain; mean prevalence was 6.1% for F. tularensis and 51% for Bartonella spp. Contrasted vector-host associations in the prevalence of these bacteria suggest that fleas have distinct roles in the transmission cycle of each pathogen in nature.

Arthropod-Borne Bacteria Cause Nonmalarial Fever in Rural Ethiopia: A Cross-Sectional Study in 394 Patients.

Bacterial arthropod-borne pathogens are a common cause of fever in Africa, but their precise impact is unknown and usually underdiagnosed in the basic rural laboratories of low-resourced African countries. Our aim was to determine the prevalence of arthropod-borne bacterial diseases causing fever among malaria smear-negative patients in a rural hospital located in Ethiopia. The study population included patients aged 2 years or older; referred to Gambo Rural General Hospital (West Arsi, Ethiopia), between July and November 2013, for fever or report of fever in the previous 48 h; attending the outpatient department; and testing negative for malaria by Giemsa-stained thin blood smears. We extracted DNA from 394 whole blood samples, using reverse line blot assays of amplicons to look for bacteria from the genera: Anaplasma, Bartonella, Borrelia, Coxiella, Ehrlichia, Francisella, and Rickettsia. Thirteen patients showed presence of DNA for these pathogens: three each by Borrelia spp., the Francisella group (F. tularensis tularensis, F. tularensis holartica, and F. novicia), Rickettsia bellii, and Rickettsia Felis, and one by Bartonella rochalimae. Thus, in this rural area of Africa, febrile symptoms could be due to bacteria transmitted by arthropods. Further studies are needed to evaluate the pathogenic role of R. bellii.

Una causa inesperada de disfagia: mesotelioma pleural / An unexpected cause of dysphagia: pleural mesothelioma

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Zoonotic pathogens in fluctuating common vole (Microtus arvalis) populations: occurrence and dynamics.

Diseases and host dynamics are linked, but their associations may vary in strength, be time-lagged, and depend on environmental influences. Where a vector is involved in disease transmission, its dynamics are an additional influence, and we often lack a general understanding on how diseases, hosts and vectors interact. We report on the occurrence of six zoonotic arthropod-borne pathogens (Anaplasma, Bartonella, Borrelia, Coxiella, Francisella and Rickettsia) in common voles (Microtus arvalis) throughout a population fluctuation and how their prevalence varies according to host density, seasonality and vector prevalence. We detected Francisella tularensis and four species of Bartonella, but not Anaplasma, Borrelia, Coxiella or Rickettsia. Bartonella taylorii and B. grahamii prevalence increased and decreased with current host (vole and mice) density, respectively, and increased with flea prevalence. Bartonella doshiae prevalence decreased with mice density. These three Bartonella species were also more prevalent during winter. Bartonella rochalimae prevalence varied with current and previous vole density (delayed-density dependence), but not with season. Coinfection with F. tularensis and Bartonella occurred as expected from the respective prevalence of each disease in voles. Our results highlight that simultaneously considering pathogen, vector and host dynamics provide a better understanding of the epidemiological dynamics of zoonoses in farmland rodents.

Density-Dependent Prevalence of Francisella tularensis in Fluctuating Vole Populations, Northwestern Spain.

Tularemia in humans in northwestern Spain is associated with increases in vole populations. Prevalence of infection with Francisella tularensis in common voles increased to 33% during a vole population fluctuation. This finding confirms that voles are spillover agents for zoonotic outbreaks. Ecologic interactions associated with tularemia prevention should be considered.

Towards Development of Improved Serodiagnostics for Tularemia by Use of Francisella tularensis Proteome Microarrays.

Tularemia in humans is caused mainly by two subspecies of the Gram-negative facultative anaerobe Francisella tularensis: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B). The current serological test for tularemia is based on agglutination of whole organisms, and the reactive antigens are not well understood. Previously, we profiled the antibody responses in type A and B tularemia cases in the United States using a proteome microarray of 1,741 different proteins derived from the type A strain Schu S4. Fifteen dominant antigens able to detect antibodies to both types of infection were identified, although these were not validated in a different immunoassay format. Since type A and B subspecies are closely related, we hypothesized that Schu S4 antigens would also have utility for diagnosing type B tularemia caused by strains from other geographic locations. To test this, we probed the Schu S4 array with sera from 241 type B tularemia cases in Spain. Despite there being no type A strains in Spain, we confirmed the responses against some of the same potential serodiagnostic antigens reported previously, as well as determined the responses against additional potential serodiagnostic antigens. Five potential serodiagnostic antigens were evaluated on immunostrips, and two of these (FTT1696/GroEL and FTT0975/conserved hypothetical protein) discriminated between the Spanish tularemia cases and healthy controls. We conclude that antigens from the type A strain Schu S4 are suitable for detection of antibodies from patients with type B F. tularensis infections and that these can be used for the diagnosis of tularemia in a deployable format, such as the immunostrip.

Long-range dispersal moved Francisella tularensis into Western Europe from the East.

For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of Francisella tularensis, the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (n=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (n=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the F. tularensis population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that F. tularensis has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species.

Tularemia Outbreaks and Common Vole (Microtus arvalis) Irruptive Population Dynamics in Northwestern Spain, 1997-2014.

During the last decades, large tularemia outbreaks in humans have coincided in time and space with population outbreaks of common voles in northwestern Spain, leading us to hypothesize that this rodent species acts as a key spillover agent of Francisella tularensis in the region. Here, we evaluate for the first time a potential link between irruptive vole numbers and human tularemia outbreaks in Spain. We compiled vole abundance estimates obtained through live-trapping monitoring studies and official reports of human tularemia cases during the period 1997-2014. We confirm a significant positive association between yearly cases of tularemia infection in humans and vole abundance. High vole densities during outbreaks (up to 1000 voles/hectare) may therefore enhance disease transmission and spillover contamination in the environment. If this ecological link is further confirmed, the apparent multiannual cyclicity of common vole outbreaks might provide a basis for forecasting the risk of tularemia outbreaks in northwestern Spain.

The importance of lizards and small mammals as reservoirs for Borrelia lusitaniae in Portugal.

Borrelia lusitaniae is a pathogen frequent in the Mediterranean area. Apart from lizards, evidence for birds and small mammals as competent reservoirs for this genospecies has been occasional. We collected questing ticks, skin biopsies and Ixodes sp. ticks feeding on lizards, birds and small mammals in a B. burgdorferi s.l. (sensu lato) enzootic area to assess their importance in the maintenance of B. lusitaniae. Borrelia lusitaniae was the most prevalent genospecies in questing ticks and was commonly found in larvae feeding on Psammodromus algirus. One biopsy infected with B. lusitaniae was collected from the tail of one Podarcis hispanica, which suggests systemic infection. Ixodes ricinus larvae feeding on Apodemus sylvaticus were infected with B. lusitaniae but with a lower prevalence. Our results reinforce the importance of lizards as reservoirs for B. lusitaniae, suggesting that P. algirus, in particular, acts as main reservoir for B. lusitaniae in Portugal.

Distribution of Bartonella henselae variants in patients, reservoir hosts and vectors in Spain.

We have studied the diversity of B. henselae circulating in patients, reservoir hosts and vectors in Spain. In total, we have fully characterized 53 clinical samples from 46 patients, as well as 78 B. henselae isolates obtained from 35 cats from La Rioja and Catalonia (northeastern Spain), four positive cat blood samples from which no isolates were obtained, and three positive fleas by Multiple Locus Sequence Typing and Multiple Locus Variable Number Tandem Repeats Analysis. This study represents the largest series of human cases characterized with these methods, with 10 different sequence types and 41 MLVA profiles. Two of the sequence types and 35 of the profiles were not described previously. Most of the B. henselae variants belonged to ST5. Also, we have identified a common profile (72) which is well distributed in Spain and was found to persist over time. Indeed, this profile seems to be the origin from which most of the variants identified in this study have been generated. In addition, ST5, ST6 and ST9 were found associated with felines, whereas ST1, ST5 and ST8 were the most frequent sequence types found infecting humans. Interestingly, some of the feline associated variants never found on patients were located in a separate clade, which could represent a group of strains less pathogenic for humans.