Conspicuous Smooth and White Egg-Shaped Sulfur Structures on a Deep-Sea Hydrothermal Vent Formed by Sulfide-Oxidizing Bacteria.

Conspicuous egg-shaped, white, and smooth structures were observed at a hydrothermal vent site in the Guaymas Basin, Gulf of California. The gelatinous structures decomposed within hours after sampling. Scanning electron microscopy (SEM) and light microscopy showed that the structure consisted of filaments of less than 0.1 µm thickness, similar to those observed for "Candidatus Arcobacter sulfidicus." SEM-energy-dispersive X-ray spectroscopy (EDS) showed that the filaments were sulfur rich. According to 16S rRNA gene amplicon and fluorescence in situ hybridization (FISH) analyses, Arcobacter, a sulfide oxidizer that is known to produce filamentous elemental sulfur, was among the dominant species in the structure and was likely responsible for its formation. Arcobacter normally produces woolly snowflake like structures in opposed gradients of sulfide and oxygen. In the laboratory, we observed sulfide consumption in the anoxic zone of the structure, suggesting an anaerobic conversion. The sulfide oxidation and decomposition of the structure in the laboratory may be explained by dissolution of the sulfur filaments by reaction with sulfide under formation of polysulfides. IMPORTANCE At the deep-sea Guaymas Basin hydrothermal vent system, sulfide-rich hydrothermal fluids mix with oxygenated seawater, thereby providing a habitat for microbial sulfur oxidation. Microbial sulfur oxidation in the deep sea involves a variety of organisms and processes and can result in the excretion of elemental sulfur. Here, we report on conspicuous white and smooth gelatinous structures found on hot vents. These strange egg-shaped structures were often observed on previous occasions in the Guaymas Basin, but their composition and formation process were unknown. Our data suggest that the notable and highly ephemeral structure was likely formed by the well-known sulfide-oxidizing Arcobacter. While normally Arcobacter produces loose flocs or woolly layers, here smooth gel-like structures were found.

Novel microbiome dominated by Arcobacter during anoxic excurrent flow from an ocean blue hole in Andros Island, The Bahamas.

Andros Island, The Bahamas, composed of porous carbonate rock, has about 175 inland blue holes and over 50 known submerged ocean caves along its eastern barrier reef. These ocean blue holes can have both vertical and horizontal zones that penetrate under the island. Tidal forces drive water flow in and out of these caves. King Kong Cavern has a vertical collapse zone and a deep penetration under Andros Island that emits sulfidic, anoxic water and masses of thin, mucoid filaments ranging to meters in length and off-white turbid water during ebb flow. Our objective was to determine the microbial composition of this mucoid material and the unconsolidated water column turbidity based on the concept that they represent unique lithoautotrophic microbial material swept from the cave into the surrounding ocean. Bacterial DNA extracted from these filaments and surrounding turbid water was characterized using PCR that targeted a portion of the 16S rRNA gene. The genus Arcobacter dominated both the filaments and the water column above the cave entrance. Arcobacter nitrofigilis and Arcobacter sp. UDC415 in the mucoid filaments accounted for as much as 80% of mapped DNA reads. In the water column Arcobacter comprised from 65% to over 85% of the reads in the depth region from about 18 m to 34 m. Bacterial species diversity was much higher in surface water and in water deeper than 36 m than in the intermediate zone. Community composition indicates that ebb flow from the cavern influences the entire water column at least to within 6 m of the surface and perhaps the near surface as well.

From the Andes to the desert: 16S rRNA metabarcoding characterization of aquatic bacterial communities in the Rimac river, the main source of water for Lima, Peru.

The Rimac river is the main source of water for Lima, Peru's capital megacity. The river is constantly affected by different types of contamination including mine tailings in the Andes and urban sewage in the metropolitan area. In this work, we aim to produce the first characterization of aquatic bacterial communities in the Rimac river using a 16S rRNA metabarcoding approach which would be useful to identify bacterial diversity and potential understudied pathogens. We report a lower diversity in bacterial communities from the Lower Rimac (Metropolitan zone) in comparison to other sub-basins. Samples were generally grouped according to their geographical location. Bacterial classes Alphaproteobacteria, Bacteroidia, Campylobacteria, Fusobacteriia, and Gammaproteobacteria were the most frequent along the river. Arcobacter cryaerophilus (Campylobacteria) was the most frequent species in the Lower Rimac while Flavobacterium succinicans (Bacteroidia) and Hypnocyclicus (Fusobacteriia) were the most predominant in the Upper Rimac. Predicted metabolic functions in the microbiota include bacterial motility and quorum sensing. Additional metabolomic analyses showed the presence of some insecticides and herbicides in the Parac-Upper Rimac and Santa Eulalia-Parac sub-basins. The dominance in the Metropolitan area of Arcobacter cryaerophilus, an emergent pathogen associated with fecal contamination and antibiotic multiresistance, that is not usually reported in traditional microbiological quality assessments, highlights the necessity to apply next-generation sequencing tools to improve pathogen surveillance. We believe that our study will encourage the integration of omics sciences in Peru and its application on current environmental and public health issues.

Molecular detection and genetic characterization of Arcobacter butzleri isolated from red-footed pet tortoises suspected for Campylobacter spp. from Grenada, West Indies.

The aim of the study was to detect and genetically characterize Arcobacter butzleri in pet red-footed tortoises suspected for Campylobacter spp., using molecular techniques. A written consent from tortoise owners was obtained, after explaining the advantages of the research to tortoise owners of Grenada. Fecal samples were collected from 114 tortoises from five parishes of the country and cultured for Campylobacter spp. using selective culture techniques. A. butzleri was isolated from 4.39% of pet tortoises. Total thirteen isolates were obtained; all identified as A. butzleri by a universal and a species-specific Polymerase Chain Reaction (PCR) and direct sequencing. Genetic characterization of these isolates was performed based on Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) that generated eight different genetic fingerprints with a discriminatory power of 0.91. Campylobacter species were not detected molecularly in any of the culture-positive samples. This is the first report of infection of pet tortoises in Grenada, West Indies with A. butzleri. This study emphasizes on the risk of zoonotic transmission of A. butzleri by exotic pets, which is a serious concern for public health.

Arcobacter peruensis sp. nov., a Chemolithoheterotroph Isolated from Sulfide- and Organic-Rich Coastal Waters off Peru.

Members of the epsilonproteobacterial genus Arcobacter have been identified to be potentially important sulfide oxidizers in marine coastal, seep, and stratified basin environments. In the highly productive upwelling waters off the coast of Peru, Arcobacter cells comprised 3 to 25% of the total microbial community at a near-shore station where sulfide concentrations exceeded 20 µM in bottom waters. From the chemocline where the Arcobacter population exceeded 106 cells ml-1 and where high rates of denitrification (up to 6.5 ± 0.4 µM N day-1) and dark carbon fixation (2.8 ± 0.2 µM C day-1) were measured, we isolated a previously uncultivated Arcobacter species, Arcobacter peruensis sp. nov. (BCCM LMG-31510). Genomic analysis showed that A. peruensis possesses genes encoding sulfide oxidation and denitrification pathways but lacks the ability to fix CO2 via autotrophic carbon fixation pathways. Genes encoding transporters for organic carbon compounds, however, were present in the A. peruensis genome. Physiological experiments demonstrated that A. peruensis grew best on a mix of sulfide, nitrate, and acetate. Isotope labeling experiments further verified that A. peruensis completely reduced nitrate to N2 and assimilated acetate but did not fix CO2, thus coupling heterotrophic growth to sulfide oxidation and denitrification. Single-cell nanoscale secondary ion mass spectrometry analysis of samples taken from shipboard isotope labeling experiments also confirmed that the Arcobacter population in situ did not substantially fix CO2 The efficient growth yield associated with the chemolithoheterotrophic metabolism of A. peruensis may allow this Arcobacter species to rapidly bloom in eutrophic and sulfide-rich waters off the coast of Peru.IMPORTANCE Our multidisciplinary approach provides new insights into the ecophysiology of a newly isolated environmental Arcobacter species, as well as the physiological flexibility within the Arcobacter genus and sulfide-oxidizing, denitrifying microbial communities within oceanic oxygen minimum zones (OMZs). The chemolithoheterotrophic species Arcobacter peruensis may play a substantial role in the diverse consortium of bacteria that is capable of coupling denitrification and fixed nitrogen loss to sulfide oxidation in eutrophic, sulfidic coastal waters. With increasing anthropogenic pressures on coastal regions, e.g., eutrophication and deoxygenation (D. Breitburg, L. A. Levin, A. Oschlies, M. Grégoire, et al., Science 359:eaam7240, 2018, https://doi.org/10.1126/science.aam7240), niches where sulfide-oxidizing, denitrifying heterotrophs such as A. peruensis thrive are likely to expand.

NGSReadsTreatment - A Cuckoo Filter-based Tool for Removing Duplicate Reads in NGS Data.

The Next-Generation Sequencing (NGS) platforms provide a major approach to obtaining millions of short reads from samples. NGS has been used in a wide range of analyses, such as for determining genome sequences, analyzing evolutionary processes, identifying gene expression and resolving metagenomic analyses. Usually, the quality of NGS data impacts the final study conclusions. Moreover, quality assessment is generally considered the first step in data analyses to ensure the use of only reliable reads for further studies. In NGS platforms, the presence of duplicated reads (redundancy) that are usually introduced during library sequencing is a major issue. These might have a serious impact on research application, as redundancies in reads can lead to difficulties in subsequent analysis (e.g., de novo genome assembly). Herein, we present NGSReadsTreatment, a computational tool for the removal of duplicated reads in paired-end or single-end datasets. NGSReadsTreatment can handle reads from any platform with the same or different sequence lengths. Using the probabilistic structure Cuckoo Filter, the redundant reads are identified and removed by comparing the reads with themselves. Thus, no prerequisite is required beyond the set of reads. NGSReadsTreatment was compared with other redundancy removal tools in analyzing different sets of reads. The results demonstrated that NGSReadsTreatment was better than the other tools in both the amount of redundancies removed and the use of computational memory for all analyses performed. Available in https://sourceforge.net/projects/ngsreadstreatment/ .

Transcriptional analysis of flagellar and putative virulence genes of Arcobacter butzleri as an endocytobiont of Acanthamoeba castellanii.

Arcobacter butzleri is an emerging foodborne zoonotic pathogen that has been isolated from environmental water sources. This pathogen establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba found in environmental matrices such as soil and water. The principal aim of this study was to analyse the transcriptional pattern of flagellar (flaA-flaB-flgH-motA) and other putative virulence genes (ciaB-cadF-mviN-pldA) of A. butzleri during its interaction with A. castellanii by quantitative real-time PCR. The transcriptional analysis showed up-regulation of all genes analysed before A. butzleri became established as an endocytobiont of A. castellanii. In contrast, while A. butzleri remains an endocytobiont, a significant and sustained decrease in the transcription of all analysed genes was observed. Our findings suggest that A. butzleri requires a biphasic transcriptional pattern of flagellar and other putative virulence genes to establish an endosymbiotic relationship with A. castellanii.

Genotypic Characterization of Arcobacter spp. Isolated from Chicken Meat in Brazil.

Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii are Gram-negative pathogenic microorganisms that cause watery diarrhea and septicemia in humans. The aims of this study were to detect the presence of Arcobacter spp. in chicken meat from butcher shops in São Paulo (Brazil) and to verify their virulence genes and genotypic profiles. A total of 300 chicken cuts were analyzed. The results show the presence of Arcobacter spp. in 18.3% of samples, which were identified as A. butzleri (63.6%) and A. cryaerophilus (36.3%). All strains were positive for the virulence genes ciaB and mviN, followed by cj1349 (98%), pldA (94.4%), cadF (72.7%), tlyA (92.7%), hecA (49%), irgA (47.2%), and hecB (34.5%). These strains were subjected to single-enzyme amplified fragment length polymorphism. Nineteen genotypic profiles were obtained for A. butzleri, and 17 for A. cryaerophilus. These results confirm the presence of virulent strains of A. butzleri and A. cryaerophilus in the chicken meat in Brazil. The presence of potentially virulent strains of Arcobacter highlights a possible public health risk, particularly with respect to ingestion of undercooked foods and cross-contamination from uncooked foods during food preparation and contaminated utensils.

Isolation and molecular characterization of Arcobacter butzleri and Arcobacter cryaerophilus from the pork production chain in Brazil / Isolamento e caracterização molecular de Arcobacter butzleri e Arcobacter cryaerophilus de linhas de abate suíno e do comércio de carne no Brasil

Arcobacter is an emerging zoonotic pathogen, and the major transmission routes to humans are the handling or consumption of contaminated raw/undercooked food products of animal origin, water and seafood. The isolation and identification of Arcobacter species are not routine in clinical laboratories; therefore, its true incidence in human infections may be underestimated. The present study aimed to isolate and characterize Arcobacter from carcasses and fecal samples collected at swine slaughterhouses and from meat markets in São Paulo State, Brazil. The isolates were identified using multiplex-PCR to differentiate the species and analyzed by single-enzyme amplified fragment length polymorphism (SE-AFLP). Arcobacter spp. were isolated from 73.0% of swine carcasses, 4% of fecal samples and 10% of pork samples. A. butzleri was the most prevalent species identified, followed by A. cryaerophilus. Interestingly, the carcasses presented higher frequency of A. butzleri isolation, whereas only A. cryaerophilus was isolated from fecal samples. SE-AFLP enabled the characterization of A. butzleri and A. cryaerophilus into 51 and 63 profiles, respectively. The great genetic heterogeneity observed for both species corroborates previous reports. This study confirms the necessity for a standard isolation protocol and the improvement of molecular tools to further elucidate Arcobacter epidemiology.(AU)

Arcobacter é um patógeno zoonótico emergente e as principais formas de transmissão para humanos são a manipulação e o consumo de água ou alimentos contaminados crus ou mal cozidos. O isolamento e a identificação das espécies de Arcobacter não fazem parte da rotina dos laboratórios clínicos; dessa forma, a real incidência da infecção em humanos é subestimada. O presente estudo teve o objetivo de isolar e caracterizar Arcobacter de carcaças e amostras de fezes coletadas em dois abatedouros de suínos e de carne suína de dois açougues no Estado de São Paulo, Brasil. As estirpes foram identificadas utilizando multiplex-PCR para diferenciar as espécies e foram analisadas por polimorfismo no comprimento de fragmentos amplificados (SE-AFLP). Arcobacter spp. foi isolado de 73% das carcaças, 4% das amostras de fezes e de 10% das amostras de carne suína avaliadas. A. butzleri foi a espécie mais prevalente, seguida por A. cryaerophilus. As carcaças apresentaram a maior taxa de isolamento de A. butzleri enquanto que apenas A. cryaerophilus foi isolado das amostras de fezes. SE-AFLP possibilitou a caracterização de A. butzleri e A. cryaerophilus em 51 e 63 perfis de bandas, respectivamente. A grande heterogeneidade genética observada para ambas as espécies corrobora estudos previous. Estes resultados confirmam a necessidade de protocolos de isolamento padronizados e o aperfeiçoamento das ferramentas moleculares para aprofundar os conhecimetos sobre epidemiologia das infecções pelo gênero Arcobacter.(AU)

Isolation and molecular characterization of Arcobacter butzleri and Arcobacter cryaerophilus from the pork production chain in Brazil / Isolamento e caracterização molecular de Arcobacter butzleri e Arcobacter cryaerophilus de linhas de abate suíno e do comércio de carne no Brasil

Arcobacter is an emerging zoonotic pathogen, and the major transmission routes to humans are the handling or consumption of contaminated raw/undercooked food products of animal origin, water and seafood. The isolation and identification of Arcobacter species are not routine in clinical laboratories; therefore, its true incidence in human infections may be underestimated. The present study aimed to isolate and characterize Arcobacter from carcasses and fecal samples collected at swine slaughterhouses and from meat markets in São Paulo State, Brazil. The isolates were identified using multiplex-PCR to differentiate the species and analyzed by single-enzyme amplified fragment length polymorphism (SE-AFLP). Arcobacter spp. were isolated from 73.0% of swine carcasses, 4% of fecal samples and 10% of pork samples. A. butzleri was the most prevalent species identified, followed by A. cryaerophilus. Interestingly, the carcasses presented higher frequency of A. butzleri isolation, whereas only A. cryaerophilus was isolated from fecal samples. SE-AFLP enabled the characterization of A. butzleri and A. cryaerophilus into 51 and 63 profiles, respectively. The great genetic heterogeneity observed for both species corroborates previous reports. This study confirms the necessity for a standard isolation protocol and the improvement of molecular tools to further elucidate Arcobacter epidemiology.(AU)

Arcobacter é um patógeno zoonótico emergente e as principais formas de transmissão para humanos são a manipulação e o consumo de água ou alimentos contaminados crus ou mal cozidos. O isolamento e a identificação das espécies de Arcobacter não fazem parte da rotina dos laboratórios clínicos; dessa forma, a real incidência da infecção em humanos é subestimada. O presente estudo teve o objetivo de isolar e caracterizar Arcobacter de carcaças e amostras de fezes coletadas em dois abatedouros de suínos e de carne suína de dois açougues no Estado de São Paulo, Brasil. As estirpes foram identificadas utilizando multiplex-PCR para diferenciar as espécies e foram analisadas por polimorfismo no comprimento de fragmentos amplificados (SE-AFLP). Arcobacter spp. foi isolado de 73% das carcaças, 4% das amostras de fezes e de 10% das amostras de carne suína avaliadas. A. butzleri foi a espécie mais prevalente, seguida por A. cryaerophilus. As carcaças apresentaram a maior taxa de isolamento de A. butzleri enquanto que apenas A. cryaerophilus foi isolado das amostras de fezes. SE-AFLP possibilitou a caracterização de A. butzleri e A. cryaerophilus em 51 e 63 perfis de bandas, respectivamente. A grande heterogeneidade genética observada para ambas as espécies corrobora estudos previous. Estes resultados confirmam a necessidade de protocolos de isolamento padronizados e o aperfeiçoamento das ferramentas moleculares para aprofundar os conhecimetos sobre epidemiologia das infecções pelo gênero Arcobacter.(AU)

First isolation report of Arcobacter cryaerophilus from a human diarrhea sample in Costa Rica.

Arcobacter cryaerophilus is an emerging enteropathogen and potential zoonotic agent transmitted by food and water. In Costa Rica, this bacterium has not been associated with cases of human gastroenteritis, even though it has been isolated from farm animals, especially poultry. This paper reports the first isolation of A. cryaerophilus from a human case of bloody watery diarrhea and the virulence genes associated with this isolate.

Arcobacter: comparison of isolation methods, diversity, and potential pathogenic factors in commercially retailed chicken breast meat from Costa Rica.

Arcobacter species have been recognized as potential food- and waterborne pathogens. The lack of standardized isolation methods and the relatively scarce knowledge about their prevalence and distribution as emerging pathogens are due to the limitations in their detection and identification. This study aimed to determine the presence and the identification of Arcobacter in chicken breast samples commercially retailed in San José, Costa Rica, as well as to describe the adherence and invasive potential of the strains to human cells (HEp-2). Fifty chicken breast samples were collected from retail markets in the metropolitan area of the country. Six different isolation methodologies were applied for the isolation of Arcobacter. Isolation strategies consisted of combinations of enrichments in de Boer or Houf selective broths and subsequent isolation in blood agar (directly or with a previous passive membrane filtration step) or Arcobacter selective agar. Suspicious colonies were identified with a genus-specific PCR, whereas species-level identification was achieved with a multiplex PCR. The overall isolation frequency of Arcobacter was 56%. From the isolation strategies, the combination of enrichment in Houf selective broth followed by filtration on blood agar showed the best performance, with a sensitivity of 89% and a specificity of 84%. A total of 46 isolates were confirmed as Arcobacter with the genus-specific PCR, from which 27 (59%) corresponded to Arcobacter butzleri, 9 (19%) to Arcobacter cryaerophilus, and 10 (22%) were not identified with this multiplex PCR. Regarding the potential pathogenicity, 75% of the isolates presented adherence to HEp-2 cells, while only 22% were invasive to that cell line. All invasive strains were A. butzleri or nonidentified strains. The results show the presence of potentially pathogenic Arcobacter in poultry and recognize the importance it should receive as a potential foodborne pathogen from public health authorities.

Assessment of the prevalence and diversity of emergent campylobacteria in human stool samples using a combination of traditional and molecular methods.

This study aims to assess the diversity of campylobacteria (Campylobacter and Arcobacter) in human fecal samples from patients with diarrhea (n = 140) and asymptomatic controls (n = 116) in Chile, using a combination of traditional culture and molecular methods. The culture methods detected campylobacteria in 10.7% of the patients with diarrhea and in 1.7% of the controls. In contrast, the molecular methods detected campylobacteria more often than the traditional culture, with a prevalence of 25.7% and 5.2%, respectively. The traditional methods only recovered the species Campylobacter jejuni, Campylobacter coli, and Arcobacter butzleri, whereas the molecular methods additionally detected the emergent species Campylobacter concisus and Campylobacter ureolyticus.

Arcobacter defluvii sp. nov., isolated from sewage samples.

A study employing a polyphasic taxonomic approach was undertaken to clarify the position of 12 isolates recovered from sewage samples. These isolates were recognized as a potential novel species because a new and specific pattern was produced with the 16S rRNA-RFLP Arcobacter identification method. The sequences of the 16S rRNA gene not only supported the classification of these novel strains as members of the genus Arcobacter, but also showed that they formed a separate phylogenetic line. Strain SW28-11(T), chosen as the representative of these strains, showed 16S rRNA gene sequence similarity of 95.6 % with the closest related species Arcobacter nitrofigilis. The phylogenetic position of the novel strains was further confirmed by analysis of the housekeeping genes hsp60, rpoB and, for the first time, gyrB. The latter proved to be an excellent additional gene for establishing the phylogeny of this genus. These data, together with phenotypic characterization, revealed that this group of isolates represent a novel species of the genus Arcobacter. The name Arcobacter defluvii sp. nov., is proposed, with the type strain SW28-11(T) ( = CECT 7697(T) = LMG 25694(T)).

Bacterial 16S rRNA gene analysis revealed that bacteria related to Arcobacter spp. constitute an abundant and common component of the oyster microbiota (Tiostrea chilensis).

To explore the bacterial microbiota in Chilean oyster (Tiostrea chilensis), a molecular approach that permits detection of different bacteria, independently of their capacity to grow in culture media, was used. Bacterial diversity was assessed by analysis of both the 16S rDNA and the 16S-23S intergenic region, obtained by PCR amplifications of DNA extracted from depurated oysters. RFLP of the PCR amplified 16S rDNA showed a prevailing pattern in most of the individuals analyzed, indicating that a few bacterial species were relatively abundant and common in oysters. Cloning and sequencing of the 16S rDNA with the prevailing RFLP pattern indicated that this rRNA was most closely related to Arcobacter spp. However, analysis by the size of the amplified 16S-23S rRNA intergenic regions revealed not Arcobacter spp. but Staphylococcus spp. related bacteria as a major and common component in oyster. These different results may be caused by the absence of target for one of the primers employed for amplification of the intergenic region. Neither of the two bacteria species found in large abundance was recovered after culturing under aerobic, anaerobic, or microaerophilic conditions. This result, however, is expected because the number of bacteria recovered after cultivation was less than 0.01% of the total. All together, these observations suggest that Arcobacter-related strains are probably abundant and common in the Chilean oyster bacterial microbiota.