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An earlier report described a human case of necrotizing fasciitis (NF) caused by mixed infection with 4 Aeromonas hydrophila strains (NF1–NF4). While the NF2, NF3, and NF4 strains were clonal and possessed exotoxin A (ExoA), the NF1 strain was determined to be phylogenetically distinct, harboring a unique type 6 secretion system (T6SS) effector (TseC). During NF1 and NF2 mixed infection, only NF1 disseminated, while NF2 was rapidly killed by a contact-dependent mechanism and macrophage phagocytosis, as was demonstrated by using in vitro models. To confirm these findings, we developed 2 NF1 mutants (NF1Δ tseC and NF1Δ vasK ); vasK encodes an essential T6SS structural component. NF1 VasK and TseC were proven to be involved in contact-dependent killing of NF2 in vitro, as well as in its elimination at the intramuscular injection site in vivo during mixed infection, with overall reduced mouse mortality. ExoA was shown to have an important role in NF by both NF1- exoA (with cis exoA ) and NF2 during monomicrobial infection. However, the contribution of ExoA was more important for NF2 than NF1 in the murine peritonitis model. The NF2∆ exoA mutant did not significantly alter animal mortality or NF1 dissemination during mixed infection in the NF model, suggesting that the ExoA activity was significant at the injection site. Immunization of mice to ExoA protected animals from NF2 monomicrobial challenge, but not from polymicrobial infection because of NF2 clearance. This study clarified the roles of T6SS and ExoA in pathogenesis caused by A. hydrophila NF strains in both mouse peritonitis and NF models in monomicrobial and polymicrobial infections.

The bacterium Aeromonas salmonicida is known since long time as a major fish pathogen unable to grow at 37 °C. However, some cases of human infection by putative mesophilic A. salmonicida have been reported. The goal of the present study is to examine two clinical cases of human infection by A. salmonicida in Spain and to investigate the pathogenicity in mammals of selected mesophilic A. salmonicida strains. An evaluation of the pathogenicity in a mouse model of clinical and environmental A. salmonicida strains was performed. The genomes of the strains were sequenced and analyzed in order to find the virulence determinants of these strains. The experimental infection in mice showed a gradient in the virulence of these strains and that some of them can cause necrotizing fasciitis and tissue damage in the liver. In addition to demonstrating significant genomic diversity among the strains studied, bioinformatics analyses permitted also to shed light on crucial elements for the virulence of the strains, like the presence of a type III secretion system in the one that caused the highest mortality in the experimental infection. Clinicians and microbiologists should consider these results for the inclusion of A. salmonicida in diagnosis tests since it is now clear that some mesophilic strains are also pathogens for humans.

Arcobacter-like species are found associated with many matrices, including shellfish in marine environments. The culture media and conditions play a major role in the recovery of new Arcobacter-like species. This study was aimed to develop a culture media for isolation and enhanced growth of Arcobacter-like spp. from marine and shellfish matrices. For this purpose, 14 different Arcobacter-like spp. mostly isolated from shellfish, were grown in 24 different formulations of enrichment broths. The enrichment broths consisted of five main groups based on the organic contents (fresh oyster homogenate, lyophilized oyster either alone or in combination with other standard media), combined with artificial seawater (ASW) or 2.5% NaCl. Optical density (OD420nm) measurements after every 24 h were compared with the growth in control media (Arcobacter broth) in parallel. The mean and standard deviation were calculated for each species in each broth and statistical differences (p Arcobacter-like species growth was significantly higher in Arcobacter broth + 50% ASW and the same media supplemented with lyophilized oysters. This is the first study to have used fresh or lyophilized oyster flesh in the enrichment broth for isolation of shellfish-associated Arcobacter-like spp.

[EN] Viruses (e.g., noroviruses and hepatitis A and E virus), bacteria (e.g., Salmonella spp. and pathogenic Escherichia coli) and protozoa (e.g., Cryptosporidium parvum and Giardia intestinalis) are well-known contributors to food-borne illnesses linked to contaminated fresh produce. As agricultural irrigation increases the total amount of water used annually, reclaimed water is a good alternative to reduce dependency on conventional irrigation water sources. European guidelines have established acceptable concentrations of certain pathogens and/or indicators in irrigation water, depending on the irrigation system used and the irrigated crop. However, the incidences of food-borne infections are known to be underestimated and all the different pathogens contributing to these infections are not known. Next-generation sequencing (NGS) enables the determination of the viral, bacterial and protozoan populations present in a water sample, providing an opportunity to detect emerging pathogens and develop improved tools for monitoring the quality of irrigation water. This is a descriptive study of the virome, bacteriome and parasitome present in different irrigation water sources. We applied the same concentration method for all the studied samples and specific metagenomic approaches to characterize both DNA and RNA viruses, bacteria and protozoa. In general, most of the known viral species corresponded to plant viruses and bacteriophages. Viral diversity in river water varied over the year, with higher bacteriophage prevalences during the autumn and winter. Reservoir water contained Enterobacter cloacae, an opportunistic human pathogen and an indicator of fecal contamination, as well as Naegleria australiensis and Naegleria clarki. Hepatitis E virus and Naegleria fowleri, emerging human pathogens, were detected in groundwater. Reclaimed water produced in a constructed wetland system presented a virome and bacteriome that resembled those of freshwater samples (river and reservoir water). Viral, bacterial and protozoan pathogens were occasionally detected in the different irrigation water sources included in this study, justifying the use of improved NGS techniques to get a comprehensive evaluation of microbial species and potential environmental health hazards associated to irrigation water., This work was supported through a grant funded by the Spanish Ministry of Economy and Competitiveness (MINECO) in the frame of the collaborative international consortium JPIW2013-095-C03-01, JPIW2013-095-C03-02 and JPIW2013-095-C03-03 of the Water Challenges for a Changing World Joint Programming Initiative (Water JPI) Pilot Call and partially by AGL2017-86797-C2-1-R. Silvia Bofill-Mas is a Serra-Hunter fellow at the University of Barcelona.

[EN] The wide diversity of irrigation water sources (i.e., drinking water, groundwater, reservoir water, river water) includes reclaimed water as a requested measure for increasing water availability, but it is also a challenge as pathogen exposure may increase. This study evaluates the level of microbial contamination in different irrigation waters to improve the knowledge and analyses management measures for safety irrigation. Over a one-year period, the occurrence of a set of viruses, bacteria and protozoa, was quantified and the performance of a wetland system, producing reclaimed water intended for irrigation, was characterized. Human fecal pollution (HAdV) was found in most of the irrigation water types analysed. Hepatitis E virus (HEV), an emerging zoonotic pathogen, was present in groundwater where porcine contamination was identified (PAdV). The skin-carcinoma associated Merkel cell polyomavirus (MCPyV), was found occasionally in river water. Noroviruses were detected, as expected, in winter, in river water and reclaimed water. Groundwater, river water and reservoir water also harboured potential bacterial pathogens, like Helicobacter pylori, Legionella spp. and Aeromonas spp. that could be internalized and viable inside amoebas like Acanthamoeba castellanii, which was also detected. Neither Giardia cysts, nor any Cryptosporidium oocysts were detected. The wetland system removed 3 Log(10) of viruses and 5 Log(10) of bacteria, which resembled the river water quality. Irrigation waters were prone to variable contamination levels and according to the European guidance documents, the E. coli (EC) levels were not always acceptable. Sporadic detection of viral pathogens as NoV GII and HAdV was identified in water samples presenting lower EC than the established limit (100MNP/100 mL). When dealing with reclaimed water as a source of irrigation the analysis of some viral parameters, like HAdV during the peak irrigation period (summer and spring) or NoV during the coldest months, could complement existing water management tools based on bacterial indicators., Thiswork was partially funded by a grant fromthe Spanish Ministry of Economy and Competitiveness (MINECO) in the frame of the collaborative international consortium JPIW2013-095-C03-01, JPIW2013095-C03-02 and JPIW2013-095-C03-03 of the Water Challenges for a Changing World Joint Programming Initiative (Water JPI) Pilot Call and partially by AGL2017-86797-C2-1-R. Silvia Bofill-Mas is a SerraHunter fellow at the University of Barcelona. Special thanks to E. Fores, E. Anfruns, and X. Romero from Granollers city council, for the valuable information and help during the sampling campaign.

Purpose. The genus Arcobacter includes bacteria that are considered emergent pathogens because they can produce infections in humans and animals. The most common symptoms are bloody and non-bloody persistent diarrhea but cases with abdominal cramps without diarrhea or asymptomatic cases have also been described as well as cases with bacteremia. The objective was to characterize Arcobacter clinical strains isolated from the faeces of patients from three Spanish hospitals. Methodology. We have characterized 28 clinical strains (27 of A. butzleri and one of A. cryaerophilus) isolated from faeces, analysing their epidemiological relationship using the multilocus sequence typing (MLST) approach and screening them for their antibiotic susceptibility and for the presence of virulence genes. Results/Key findings. Typing results showed that only one of the 28 identified sequence types (i.e. ST 2) was already present in the MLST database. The other 27 STs constituted new records because they included new alleles for five of the seven genes or new combinations of known alleles of the seven genes. All strains were positive for the ciaB virulence gene and sensitive to tetracycline. However, 7.4 % of the A. butzleri and A. cryaerophilus strains showed resistance to ciprofloxacin. Conclusion. The fact that epidemiological unrelated strains show the same ST indicates that other techniques with higher resolution should be developed to effectively recognize the infection source. Resistance to ciprofloxacin, one of the antibiotics recommended for the treatment of Arcobacter intestinal infections, demonstrated in 10.7 % of the strains, indicates the importance of selecting the most appropriate effective treatment.

An earlier report described a human case of necrotizing fasciitis (NF) caused by mixed infection with 4 Aeromonas hydrophila strains (NF1–NF4). While the NF2, NF3, and NF4 strains were clonal and possessed exotoxin A (ExoA), the NF1 strain was determined to be phylogenetically distinct, harboring a unique type 6 secretion system (T6SS) effector (TseC). During NF1 and NF2 mixed infection, only NF1 disseminated, while NF2 was rapidly killed by a contact-dependent mechanism and macrophage phagocytosis, as was demonstrated by using in vitro models. To confirm these findings, we developed 2 NF1 mutants (NF1ΔtseC and NF1ΔvasK); vasK encodes an essential T6SS structural component. NF1 VasK and TseC were proven to be involved in contact-dependent killing of NF2 in vitro, as well as in its elimination at the intramuscular injection site in vivo during mixed infection, with overall reduced mouse mortality. ExoA was shown to have an important role in NF by both NF1-exoA (with cis exoA) and NF2 during monomicrobial infection. However, the contribution of ExoA was more important for NF2 than NF1 in the murine peritonitis model. The NF2∆exoA mutant did not significantly alter animal mortality or NF1 dissemination during mixed infection in the NF model, suggesting that the ExoA activity was significant at the injection site. Immunization of mice to ExoA protected animals from NF2 monomicrobial challenge, but not from polymicrobial infection because of NF2 clearance. This study clarified the roles of T6SS and ExoA in pathogenesis caused by A. hydrophila NF strains in both mouse peritonitis and NF models in monomicrobial and polymicrobial infections.

The genus Aeromonas belongs to the Aeromonadaceae family and comprises a group of Gram-negative bacteria widely distributed in aquatic environments, with some species able to cause disease in humans, fish, and other aquatic animals. However, bacteria of this genus are isolated from many other habitats, environments, and food products. The taxonomy of this genus is complex when phenotypic identification methods are used because such methods might not correctly identify all the species. On the other hand, molecular methods have proven very reliable, such as using the sequences of concatenated housekeeping genes like gyrB and rpoD or comparing the genomes with the type strains using a genomic index, such as the average nucleotide identity (ANI) or in silico DNA–DNA hybridization (isDDH). So far, 36 species have been described in the genus Aeromonas of which at least 19 are considered emerging pathogens to humans, causing a broad spectrum of infections. Having said that, when classifying 1852 strains that have been reported in various recent clinical cases, 95.4% were identified as only four species: Aeromonas caviae (37.26%), Aeromonas dhakensis (23.49%), Aeromonas veronii (21.54%), and Aeromonas hydrophila (13.07%). Since aeromonads were first associated with human disease, gastroenteritis, bacteremia, and wound infections have dominated. The literature shows that the pathogenic potential of Aeromonas is considered multifactorial and the presence of several virulence factors allows these bacteria to adhere, invade, and destroy the host cells, overcoming the immune host response. Based on current information about the ecology, epidemiology, and pathogenicity of the genus Aeromonas, we should assume that the infections these bacteria produce will remain a great health problem in the future. The ubiquitous distribution of these bacteria and the increasing elderly population, to whom these bacteria are an opportunistic pathogen, will facilitate this problem. In addition, using data from outbreak studies, it has been recognized that in cases of diarrhea, the infective dose of Aeromonas is relatively low. These poorly known bacteria should therefore be considered similarly as enteropathogens like Salmonella and Campylobacter.

Metagenomic analysis of virus, bacteria and protozoa in irrigation water.

During a survey in a dairy plant in Italy, the second strain (strain FG 206) of Arcobacter suis described in the literature was isolated from raw water buffalo milk. The objective of this study was to confirm the species identification, better define the species by comparing its characteristics with those of the reference strain (F41 T = CECT 7833 T = LMG 26152 T ) and to investigate its potential clinical relevance by detecting the virulence gene pattern of the new strain. Phenotypical characterization and 16S rRNA-RFLP gave a complete overlap of results for the two strains. As expected, an RFLP pattern common to A. suis and Arcobacter defluvii was obtained by MseI endonuclease digestion, and a pattern specific for A. suis was obtained by BfaI endonuclease digestion. 16S rRNA sequencing and multilocus phylogenetic analysis (MLPA) showed a robust relatedness of strain FG 206 to the A. suis type strain F41 T . The recovery of strain FG 206 from a dairy plant shows that this species of Arcobacter is present in the food chain. Like the type strain recovered from pig meat, the species A. suis may not be confined to a single type of food.

To perform a comparative study for determining the optimum culture method (direct plating or enrichment) and medium (ampicillin dextrin agar (ADA), starch ampicillin agar (SAA), bile salts irgasan brilliant green modified (BIBG-m)) for recovering Aeromonas species from water and shellfish samples.By direct culture, Aeromonas was detected in 65% (13/20) of the water samples and in 54·5% (6/11) of the shellfish samples. However, when a pre-enrichment step was included, the number of positive water samples increased to 75% (15/20) and the ones of shellfish to 90·1% (10/11). The enriched culture significantly favoured (P 0·05) the isolation of Aeromonas allosaccharophila from water, Aeromonas salmonicida from shellfish and Aeromonas caviae from both types of samples. The most specific (P 0·05) culture medium for detecting Aeromonas from water was ADA. However, no differences were observed in the case of shellfish samples (P 0·05). Isolation of Aeromonas media from water was favoured (P 0·05) in the ADA medium, while SAA enhanced (P 0·05) the isolation of Aer. salmonicida from shellfish.The culture method and medium used influenced the recovery of some Aeromonas species from water and shellfish samples.This fact should be considered in future prevalence studies to avoid overestimating the above mentioned Aeromonas species.

The use of lagooning as a complementary natural method of treating secondary effluents of wastewater treatment plants has been employed as an affordable and easy means of producing reclaimed water. However, using reclaimed water for some purposes, for example, for food irrigation, presents some risks if the effluents contain microbial pathogens. Classical bacterial indicators that are used to assess faecal contamination in water do not always properly indicate the presence of bacterial or viral pathogens. In the current study, the presence of faecal indicator bacteria (FIB), heterotrophic bacterial counts (HBC), pathogens and opportunistic pathogens, such as Legionella spp., Aeromonas spp., Arcobacter spp., free-living amoeba (FLA), several viral indicators (human adenovirus and polyomavirus JC) and viral pathogens (noroviruses and hepatitis E virus) were analysed for 1 year in inlet and outlet water to assess the removal efficiency of a lagooning system. We observed 2.58 (1.17-4.59) and 1.65 (0.15-3.14) log reductions in Escherichia coli (EC) and intestinal enterococci (IE), respectively, between the inlet and outlet samples. Genomic copies of the viruses were log reduced by 1.18 (0.24-2.93), 0.64 (0.12-1.97), 0.45 (0.04-2.54) and 0.72 (0.22-2.50) for human adenovirus (HAdV), JC polyomavirus (JCPyV) and human noroviruses (NoV GI and GII), respectively. No regrowth of opportunistic pathogens was observed within the system. FLA, detected in all samples, did not show a clear trend. The reduction of faecal pathogens was irregular with 6 out of 12 samples and 4 out of 12 samples exceeding the EC and IE values, specified in the Spanish legislation for reclaimed water (RD 1620/2007). This data evidences that there is a need for more studies to evaluate the removal mechanisms of lagooning systems in order to optimize pathogen reduction. Moreover, surveillance of water used to irrigate raw edible vegetables should be conducted to ensure the fulfilment of the microbial requirements for the production of safe reclaimed water.

Two isolates, one recovered from a carrot and another one from urban wastewater, were characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both isolates clustered together, and were most closely related to Aliarcobacter lanthieri. Multilocus phylogenetic analysis (MLPA) using the concatenated sequences of five housekeeping genes (atpA, gyrA, gyrB, hsp60 and rpoB) suggested that these isolates formed a distinct phylogenetic lineage among the genera derived from the former genus Arcobacter. Whole-genome sequence, in silico DNA-DNA hybridization (isDDH) and the average nucleotide identity (ANI) value between the genome of strain F199T and those of related species confirmed that these isolates represent a novel species. These strains can be differentiated from its phylogenetically closest species A. lanthieri by its inability to growth on 1% glycine and by their enzyme activity of esterase lipase (C8) and acid phosphatase. Our results, by the application of a polyphasic analysis, confirmed that these two isolates represent a novel species of the genus Aliarcobacter, for which the name Aliarcobacter vitoriensis sp. nov. is proposed. The type strain is F199T (=CECT 9230T=LMG 30050T).

Post-print (lokagerð höfundar), Drinking water quality has been regulated in most European countries for nearly two decades by the drinking water directive 98/83/EC. The directive is now under revision with the goal of meeting stricter demands for safe water for all citizens, as safe water has been recognized as a human right by the United Nations. An important change to the directive is the implementation of a risk-based approach in all regulated water supplies. The European Union Framework Seventh Programme Aquavalens project has developed several new detection technologies for pathogens and indicators and tested them in water supplies in seven European countries. One of the tasks of the project was to evaluate the impact of these new techniques on water safety and on water safety management. Data were collected on risk factors to water safety for five large supplies in Denmark, Germany, Spain and the UK, and for fifteen small water supplies in Scotland, Portugal and Serbia, via a questionnaire aiming to ascertain risk factors and the stage of implementation of Water Safety Plans, and via site-specific surveys known as Sanitary Site Inspection. Samples were collected from the water supplies from all stages of water production to delivery. Pathogens were detected in around 23% of the 470 samples tested. Fecal contamination was high in raw water and even in treated water at the small supplies. Old infrastructure was considered a challenge at all the water supplies. The results showed that some of the technique, if implemented as part of the water safety management, can detect rapidly the most common waterborne pathogens and fecal pollution indicators and therefore have a great early warning potential; can improve water safety for the consumer; can validate whether mitigation methods are working as intended; and can confirm the quality of the water at source and at the tap., This work was supported by the European Union Seventh Framework Programme under the AQUAVALENS project (Grant Agreement number 311846).

The draft whole-genome sequence of Arcobacter haliotis strain LMG 28652T was obtained and compared against the type strain of Arcobacter lekithochrous LFT 1.7T. High similarity was found between the two strains, showing average nucleotide identity and in silico DNA–DNA hybridization values of 98.40 and 86.10 %, respectively. These values indicated that both genomes belonged to the same species, confirming the evidences derived from the phylogenetic analysis performed with the 16S rRNA gene and the concatenated sequences of five housekeeping genes. In addition, the metabolic, physiological and chemotaxonomic features of A. haliotis LMG 28652T were shown to be congruent with those of A. lekithochrous . We conclude that Arcobacter haliotis Tanaka et al. 2017 is a later heterotypic synonym of Arcobacter lekithochrous Dieguez et al. 2017.

Conventional wastewater treatment does not completely remove and/or inactive viruses; consequently, viruses excreted by the population can be detected in the environment. This study was undertaken to investigate the distribution and seasonality of human viruses and faecal indicator bacteria (FIB) in a river catchment located in a typical Mediterranean climate region and to discuss future trends in relation to climate change. Sample matrices included river water, untreated and treated wastewater from a wastewater treatment plant within the catchment area, and seawater from potentially impacted bathing water. Five viruses were analysed in the study. Human adenovirus (HAdV) and JC polyomavirus (JCPyV) were analysed as indicators of human faecal contamination of human pathogens; both were reported in urban wastewater (mean values of 10 6 and 10 5 GC/L, respectively), river water (10 3 and 10 2 GC/L) and seawater (10 2 and 10 1 GC/L). Human Merkel Cell polyomavirus (MCPyV), which is associated with Merkel Cell carcinoma, was detected in 75% of the raw wastewater samples (31/37) and quantified by a newly developed quantitative polymerase chain reaction (qPCR) assay with mean concentrations of 10 4 GC/L. This virus is related to skin cancer in susceptible individuals and was found in 29% and 18% of river water and seawater samples, respectively. Seasonality was only observed for norovirus genogroup II (NoV GGII), which was more abundant in cold months with levels up to 10 4 GC/L in river water. Human hepatitis E virus (HEV) was detected in 13.5% of the wastewater samples when analysed by nested PCR (nPCR). Secondary biological treatment (i.e., activated sludge) and tertiary sewage disinfection including chlorination, flocculation and UV radiation removed between 2.22 and 4.52 log 10 of the viral concentrations. Climate projections for the Mediterranean climate areas and the selected river catchment estimate general warming and changes in precipitation distribution. Persistent decreases in precipitation during summer can lead to a higher presence of human viruses because river and sea water present the highest viral concentrations during warmer months. In a global context, wastewater management will be the key to preventing environmental dispersion of human faecal pathogens in future climate change scenarios.

We performed a screening investigation to assess the human health risks of the Integrated Waste Management Facility (IWMF: mechanical-biological treatment (MBT) plant plus municipal solid waste incinerator (MSWI); Ecoparc-3) of Barcelona (Spain). Air concentrations of pollutants potentially released by the MBT plant (VOCs and bioaerosols) and the MSWI (trace elements, PCDD/Fs and PCBs) were determined. Trace elements, PCDD/Fs and PCBs were also analyzed in soil samples. The concentrations of trace elements and bioaerosols were similar to those previously reported in other areas of similar characteristics, while formaldehyde was the predominant VOC. Interestingly, PCDD/F concentrations in soil and air were the highest ever reported near a MSWI in Catalonia, being maximum concentrations 10.8 ng WHO-TEQ/kg and 41.3 fg WHO-TEQ/m(3), respectively. In addition, there has not been any reduction in soils, even after the closure of a power plant located adjacently. Human health risks of PCDD/F exposure in the closest urban nucleus located downwind the MSWI are up to 10-times higher than those nearby other MSWIs in Catalonia. Although results must be considered as very preliminary, they are a serious warning for local authorities. We strongly recommend to conduct additional studies to confirm these findings and, if necessary, to implement measures to urgently mitigate the impact of the MSWI on the surrounding environment. We must also state the tremendous importance of an individual evaluation of MSWIs, rather than generalizing their environmental and health risks.

Three groups of Aeromonas strains isolated from Finland lakes experiencing cyanobacterial blooms could not be assigned to any known species of this genus on the basis of 16S rRNA and rpoD gene sequences. The Multilocus Phylogenetic Analysis (MLPA) of the concatenated sequence of seven genes (gyrB, rpoD, recA, dnaJ, gyrA, dnaX and atpD; 4093bp) showed that the three groups of strains did not cluster with any known Aeromonas spp. and formed three independent lineages. This was confirmed by performing the analysis with their closest relatives using 15 genes (the latter 7 and cpn60, dnaK, gltA, mdh, radA, rpoB, tsf, zipA; 8751bp). Furthermore, ANI results between the genomes of the type strains of the three potential new species and those of their close relatives were all

Arcobacter thereius is a species associated with human disease. A group of A. thereius pork strains (represented by strain LMG 24487) clustered separately from the type strain (LMG 24486 T ) in the 16S rRNA and multilocus phylogenetic trees. In silico DNA-DNA hybridization and average nucleotide identity results between their genomes (93.3 and 51.1%) confirmed ‘ Arcobacter porcinus ' (LMG 24487 T ) as a new species.

International audience; Aeromonas media is an opportunistic pathogen for human and animals mainly found in aquatic habitats and which has been noted for significant genomic and phenotypic heterogeneities. We aimed to better understand the population structure and diversity of strains currently affiliated to A. media and the related species A. rivipollensis. Forty-one strains were included in a population study integrating, multilocus genetics, phylogenetics, comparative genomics, as well as phenotypics, lifestyle, and evolutionary features. Sixteen gene-based multilocus phylogeny delineated three clades. Clades corresponded to different genomic groups or genomospecies defined by phylogenomic metrics ANI (average nucleotide identity) and isDDH (in silico DNA-DNA hybridization) on 14 whole genome sequences. DL-lactate utilization, cefoxitin susceptibility, nucleotide signatures, ribosomal multi-operon diversity, and differences in relative effect of recombination and mutation (i.e., in evolution mode) distinguished the two species Aeromonas media and Aeromonas rivipollensis. The description of these two species was emended accordingly. The genome metrics and comparative genomics suggested that a third clade is a distinct genomospecies. Beside the species delineation, genetic and genomic data analysis provided a more comprehensive knowledge of the cladogenesis determinants at the root and inside A. media species complex among aeromonads. Particular lifestyles and phenotypes as well as major differences in evolution modes may represent putative factors associated with lineage emergence and speciation within the A. media complex. Finally, the integrative and populational approach presented in this study is considered broadly in order to conciliate the delineation of taxonomic species and the population structure in bacterial genera organized in species complexes.

It is widely recognized that Aeromonas infections produce septicaemia, and ulcerative and haemorrhagic diseases in fish, causing significant mortality in both wild and farmed freshwater and marine fish species that damage the economics of the aquaculture sector. The descriptions of the complete genomes of Aeromonas species have allowed the identification of an important number of virulence genes that affect the pathogenic potential of these bacteria. This review will focus on the most relevant information derived from the available Aeromonas genomes in relation to virulence and on the diverse virulence factors that actively participate in host adherence, colonization and infection, including structural components, extracellular factors, secretion systems, iron acquisition and quorum sensing mechanisms.

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Cases of Aeromonas diarrhea have been described all over the world. The genus Aeromonas includes ca. 30 species, of which 10 have been isolated in association with gastroenteritis. The dominating species that account for ca. 96% of the identified strains are Aeromonas caviae, Aeromonas veronii, Aeromonas dhakensis and Aeromonas hydrophila. However, the role of Aeromonas as a true enteropathogen has been questioned on the basis of the lack of outbreaks, the non-fulfilment of Koch’s postulates and the low numbers of acute illnesses in the only existing human challenge study. In the present study we reassess the enteropathogenicity of Aeromonas using dose response models for microbial infection and acute illness. The analysis uses the data from the human challenge study and additional data from selected outbreak investigations where the numbers exposed and the dose were reported, allowing their inclusion as natural experiments. In the challenge study several cases of asymptomatic shedding were found (26.3%, 15/57), however, only 3.5% (2/57) of those challenged with Aeromonas developed acute enteric symptoms (i.e. diarrhea). The natural experiments showed a much higher risk of illness associated with exposure to Aeromonas, even at moderate to low doses. The median dose required for 1% illness risk, was ~1.4x104 times higher in the challenge study (1.24x104 cfu) compared to natural exposure events (0.9 cfu). The dose response assessment presented in this study shows that the combined challenge and outbreak data are consistent with high infectivity of Aeromonas, and a wide range of susceptibility to acute enteric illness. To illustrate the outcomes, we simulate the risk associated with concentrations of Aeromonas found in different water and food matrices, indicating the disease burden potentially associated with these bacteria. In conclusion this study showed that Aeromonas is highly infectious, and that human susceptibility to illness may be high, similar to undisputed enteropathogens like Campylobacter or Salmonella.

The ubiquitous “jack-of-all-trades,” Aeromonas hydrophila, is a freshwater, Gram-negative bacterial pathogen under revision in regard to its phylogenetic and functional affiliation with other aeromonads. While virulence factors are expectedly diverse across A. hydrophila strains and closely related species, our mechanistic knowledge of the vast majority of these factors is based on the molecular characterization of the strains A. hydrophila AH-3 and SSU, which were reclassified as A. piscicola AH-3 in 2009 and A. dhakensis SSU in 2013. Individually, these reclassifications raise important questions involving the applicability of previous research on A. hydrophila virulence mechanisms; however, this issue is exacerbated by a lack of genomic data on other research strains. Collectively, these changes represent a fundamental gap in the literature on A. hydrophila and confirm the necessity of biochemical, molecular, and morphological techniques in the classification of research strains that are used as a foundation for future research. This review revisits what is known about virulence in A. hydrophila and the feasibility of using comparative genomics in light of this phylogenetic revision. Conflicting data between virulence factors, secretion systems, quorum sensing, and their effect on A. hydrophila pathogenicity appears to be an artifact of inappropriate taxonomic comparisons and/or be due to the fact that these properties are strain-specific. This review audits emerging data on dominant virulence factors that are present in both A. dhakensis and A. hydrophila in order to synthesize existing data with the aim of locating where future research is needed.

Aeromonas media is an opportunistic pathogen for human and animals mainly found in aquatic habitats and which has been noted for significant genomic and phenotypic heterogeneities. We aimed to better understand the population structure and diversity of strains currently affiliated to A. media and the related species A. rivipollensis. Forty-one strains were included in a population study integrating, multilocus genetics, phylogenetics, comparative genomics, as well as phenotypics, lifestyle, and evolutionary features. Sixteen gene-based multilocus phylogeny delineated three clades. Clades corresponded to different genomic groups or genomospecies defined by phylogenomic metrics ANI (average nucleotide identity) and isDDH (in silico DNA-DNA hybridization) on 14 whole genome sequences. DL-lactate utilization, cefoxitin susceptibility, nucleotide signatures, ribosomal multi-operon diversity, and differences in relative effect of recombination and mutation (i.e., in evolution mode) distinguished the two species Aeromonas media and Aeromonas rivipollensis. The description of these two species was emended accordingly. The genome metrics and comparative genomics suggested that a third clade is a distinct genomospecies. Beside the species delineation, genetic and genomic data analysis provided a more comprehensive knowledge of the cladogenesis determinants at the root and inside A. media species complex among aeromonads. Particular lifestyles and phenotypes as well as major differences in evolution modes may represent putative factors associated with lineage emergence and speciation within the A. media complex. Finally, the integrative and populational approach presented in this study is considered broadly in order to conciliate the delineation of taxonomic species and the population structure in bacterial genera organized in species complexes.

Given that changes in consumer food behaviours have led to an increase in the demand for pre-cut ready-to-eat (RTE) vegetables, and that few data are currently available on the occurrence of Arcobacter spp. in such foods, the aim of the present study was to assess the occurrence of Arcobacter spp. that carry virulence-associated genes on pre-cut RTE vegetables, using cultural and molecular methods. Arcobacter was detected using biomolecular identification methods in 44/160 (27.5%) of the samples, of which 40/44 (90.9%) isolates corresponded to A. butzleri and 4/44 (9.1%) to A. cryaerophilus. Studying the incidence of 9 virulence-associated genes revealed the widespread distribution of these genes among the Arcobacter isolates tested. The results obtained in our research provided plenty of information on the health risks associated with the direct consumption of raw vegetables, and highlight the need to implement further studies at each level of the production chain, in order to obtain further information to help protect human health.

The genus Arcobacter includes species considered emerging food and waterborne pathogens. Despite Arcobacter has been linked to the presence of faecal pollution, few studies have investigated its prevalence in wastewater, and the only isolated species were Arcobacter butzleri and Arcobacter cryaerophilus. This study aimed to establish the prevalence of Arcobacter spp. at a WWTP using in parallel two culturing methods (direct plating and culturing after enrichment) and a direct detection by m-PCR. In addition, the genetic diversity of the isolates was established using the ERIC-PCR genotyping method. Most of the wastewater samples (96.7%) were positive for Arcobacter and a high genetic diversity was observed among the 651 investigated isolates that belonged to 424 different ERIC genotypes. However, only few strains persisted at different dates or sampling points. The use of direct plating in parallel with culturing after enrichment allowed recovering the species A. butzleri, A. cryaerophilus, Arcobacter thereius, Arcobacter defluvii, Arcobacter skirrowii, Arcobacter ellisii, Arcobacter cloacae, and Arcobacter nitrofigilis, most of them isolated for the first time from wastewater. The predominant species was A. butzleri, however, by direct plating predominated A. cryaerophilus. Therefore, the overall predominance of A. butzleri was a bias associated with the use of enrichment.

The genus Aeromonas is present in a wide variety of water environments and is recognized as potentially pathogenic to humans and animals. Members of this genus are often confused with Vibrio when using automated, commercial identification systems that are culture-dependent. This study describes a PCR detection method for Aeromonas that is cultureindependent and that targets the gcat (Glycerophospholopid-Cholesterol Acyltransferase) gene, which is specific for this genus. The GCAT-PCR was 100% specific in artificially inoculated water samples, with a detection limit that ranged from 2.5 to 25 cfu/mL. The success at detecting this pathogen in 86 water samples using the GCAT-PCR method was identical to the conventional culturing method when a pre-enrichment step was carried out, yielding 83.7% positive samples. On the other hand, without a pre-enrichment step, only 77.9% of the samples were positive by culturing and only 15.1% with the GCAT-PCR. However, 83.7% positive samples were obtained for the GCAT-PCR when the water volume for the DNA extraction was increased from 400 μL to 4 mL. The proposed molecular method is much faster (5 or 29 h) than the culturing method (24 or 48 h) whether performed directly or after a pre-enrichment step and it will enable the fast detection of Aeromonas in water samples helping to prevent a possible transmission to humans.

The genusArcobacterincludes species considered emerging food and waterborne pathogens. DespiteArcobacterhas been linked to the presence of faecal pollution, few studies have investigated its prevalence in wastewater, and the only isolated species wereArcobacter butzleriandArcobacter cryaerophilus. This study aimed to establish the prevalence ofArcobacterspp. at a WWTP using in parallel two culturing methods (direct plating and culturing after enrichment) and a direct detection by m-PCR. In addition, the genetic diversity of the isolates was established using the ERIC-PCR genotyping method. Most of the wastewater samples (96.7%) were positive forArcobacterand a high genetic diversity was observed among the 651 investigated isolates that belonged to 424 different ERIC genotypes. However, only few strains persisted at different dates or sampling points. The use of direct plating in parallel with culturing after enrichment allowed recovering the species A.butzleri, A.cryaerophilus,Arcobacter thereius,Arcobacter defluvii,Arcobacter skirrowii,Arcobacter ellisii,Arcobacter cloacae, andArcobacter nitrofigilis, most of them isolated for the first time from wastewater. The predominant species was A.butzleri, however, by direct plating predominated A.cryaerophilus. Therefore, the overall predominance of A.butzleriwas a bias associated with the use of enrichment.

A re-evaluation of the taxonomic position of five strains, one assigned to Cronobacter sakazakii (strain 1330T, isolated from spiced meat purchased in Slovakia), two previously assigned to Cronobacter genomospecies 1 (strains NCTC 9529T and 731, isolated from water and a leg infection, respectively) and two previously assigned to Cronobacter turicensis (strains 96 and 1435, isolated from onion powder and rye flour, respectively) was carried out. The analysis included phenotypic characterization, 16S rRNA gene sequencing and multilocus sequence analysis (MLSA) of seven housekeeping genes (atpD, fusA, glnS, gltB, gyrB, infB, ppsA; 3036 bp). 16S rRNA gene sequence analysis and MLSA showed that strain 1330T formed an independent phylogenetic lineage in the MLSA, with Cronobacter dublinensis LMG 23823T as the closest neighbour. DNA–DNA reassociation and phenotypic analysis revealed that strain 1330T represented a novel species, for which the name Cronobacter condimenti sp. nov. is proposed (type strain 1330T = CECT 7863T = LMG 26250T). Strains NCTC 9529T, 731, 96 and 1435 clustered together within an independent phylogenetic lineage, with C. turicensis LMG 23827T as the closest neighbour in the MLSA. DNA–DNA reassociation and phenotypic analysis confirmed that these strains represent a novel species, for which the name Cronobacter universalis sp. nov. is proposed (type strain NCTC 9529T = CECT 7864T = LMG 26249T).

A group of ten Arcobacter isolates (Gram negative, slightly curved motile rods, oxidase positive) was recovered from mussels (nine) and from clams (one). These isolates could not be assigned to any known species using the molecular identification methods specific for this genus (16S rDNA-RFLP and m-PCR). The aim of this study is to establish the taxonomic position of these isolates. The 16S rRNA gene sequence similarity of mussel strain F4 T to the type strains of all other Arcobacter species ranged from 91.1% to 94.8%. The species most similar to the clams' strain F67-11 T were Arcobacter defluvii (CECT 7697 T , 97.1%) and Arcobacter ellisii (CECT 7837 T , 97.0%). On the basis of phylogenetic analyses with 16S rRNA, rpoB, gyrB and hsp60 genes, the mussel and clam strains formed two different, new lineages within the genus Arcobacter. These data, together with their different phenotypic characteristics and MALDI-TOF mass spectra, revealed that these strains represent two new species, for which the names Arcobacter bivalviorum (type strain F4 T =CECT 7835 T =LMG 26154 T ) and Arcobacter venerupis (type strain F67-11 T =CECT 7836 T =LMG 26156 T ) are proposed. © 2012 Elsevier GmbH.