Whole-Genome Sequence of Aeromonas spp. Isolated from a Dairy Farm in Central Texas

: This study investigated the presence of Aeromonas spp. on a dairy farm in central Texas that employed a free-stall management system. A total of 140 samples were collected from areas of two different barns. Twenty-two presumptive Aeromonas isolates were cultured. Phenotypic analysis identified five Aeromonas spp. Twenty isolates exhibited β-lactam and one displayed tetracycline resistance. Phylogenetic analysis of the WGS data suggested only four Aeromonas spp. All isolates possessed at least one β-lactam resistance gene and one isolate possessed tet (E). No plasmids were identified from sequence alignments. Virulence genes were identified in all four Aeromonas spp. Mobility elements were identified in three of these, with the exception being A. dhakensis . Four of the transposons identified in this study have been associated with multidrug resistance in Italy, Sweden, and Singapore. There was no significant difference in the proportion of isolates from either barn. The absence of plasmids suggests mobility elements and virulence genes were localized to the chromosome. On a dairy farm of healthy cattle, these 22 Aeromonas isolates were considered normal environmental flora while illustrating the ubiquitous nature of Aeromonas spp. globally.


Introduction
Aeromonas are currently comprised of 36 bacterial species and 12 subspecies.Taxonomically, the identification of the genus and species of Aeromonads has been constantly in flux due to differences in phenotypic and genotypic profiles [1,2].Aeromonas are autochthonous in aquatic environments ranging from marine to freshwater, and from water treatment plants to raw sewage [3][4][5].Aeromonads provide an excellent example of the importance of "One Health", where antimicrobial-resistant and pathogenic bacteria intersect the interface of environment, healthcare, and agriculture.Such interactions provide the opportunity for the dissemination of pathogenic bacteria or undesirable genetic elements to occur.There are efforts to mitigate the dissemination of antimicrobial resistance, virulence, and mobile genetic elements among human, animal, and environmental reservoirs and Aeromonas should be included in this effort [6,7].
It has been known for decades that Aeromonads cause disease in fish and amphibians.Aeromonads have been less frequently isolated from humans, food production animals, domesticated pets, invertebrate species, birds, ticks, insects, and soil [3,[8][9][10].Because they can grow at lower temperatures, Aeromonas spp.have been isolated from fresh groceries stored at 4 °C, such as dairy, beef, pork, poultry products, and packaged ready-to-eat meats [11][12][13].The presence of Aeromonads in diverse niches results in shared microbial communities where they could potentially cause illness in susceptible species or transfer mobile genetic elements to other bacterial genera.A. hydrophila, A. caviae, A. veronii, and A. dhakensis are the most significant species with regard to human health [1,14].They are considered emerging pathogens and have been identified as the etiologic agent in a variety of human infections in both immunocompetent and immunocompromised humans [15][16][17].Severe infections include septicemia, peritonitis, endocarditis, gastroenteritis, and wound infections [18].Aeromonas spp.have also caused hemolytic-uremic syndrome and necrotizing fasciitis among humans [16,19].
Currently, Aeromonas infections are not common in human clinical medicine [20].Nevertheless, the potential severity of illness is an important reason they should be correctly identified as early as possible.Aeromonas spp.are Gram-negative bacilli that are oxidase-, indole-, and catalase-positive and that ferment glucose [1,15].A motile mesophilic group, typified by A. hydrophila, are associated with human disease and achieve optimal growth between 35 and 37 °C.A nonmotile psychrophilic group, typified by A. salmonicida, is associated with fish diseases and achieves optimal growth at temperatures between 22 and 25 °C [2,10].
Pathogenesis by Aeromonas spp.involves multiple metabolic factors, many of which were identified as belonging to A. hydrophila but are now known to also belong to other Aeromonas spp.[30].Regardless of species, Aeromonas spp.possess a number of known virulence genes: arylamidases, esterases, amylase, elastase, chitinases, lipases, peptidases, hydrolases, and others, that play roles in their pathogenesis [10,20,[31][32][33].Understanding the interplay between antimicrobial resistance and virulence elements is increasingly important, as many strains have acquired multiple mobile genetic elements that carry these genes among genera [16,34,35].Acquisition of mobile genetic elements does not have to be limited to one event; strains may acquire multiple resistance or virulence genes, particularly when selection pressure exists.Multiple genetic acquisitions have resulted in multidrug-resistant bacteria in many genera that can transfer mobile genetic elements into a diversity of bacteria [29,33].One multidrug-resistant A. hydrophila strain was found in a food production farm among neonatal swine with diarrhea [34,36].
With that in mind, this project was undertaken to assess the occurrence of mobility elements and virulence genes in Aeromonas spp.isolated from a working dairy farm with healthy adult Holstein cows.A dairy farm with two ventilation systems was chosen to determine if there was a difference in the prevalence of Aeromonas spp. between management systems.

Sample Collection
Samples were collected in June 2018 from a single free-stall dairy farm in central Texas that maintained approximately 1000 healthy Holstein cattle.Housing systems employed two ventilation systems: (1) flow-through or (2) cross-ventilation.The flowthrough or cross-ventilation housing structures were parallel to each other with roof peaks perpendicular to prevailing summer winds.Side curtain walls could be lowered to protect cows from morning or afternoon sun.Fans were present to aid ventilation.At the time of collection, barn curtains were up and air could flow throughout the entire facility.All cows were in open stanchions during hay consumption.Water misters were employed for cooling when necessary.All water troughs contained chilled circulated clean water; some sediment was present on the bottom of water troughs.Cows were free to mill around or lay down on clean sand floors.Cows were not in proximity to the two sump ponds that were in use at the time of sample collection.
A total of 140 samples were collected in sterile specimen jars from water troughs, barn aisles, and sump ponds from the flow-through and the cross-ventilated barns.All samples were aqueous in nature and were scooped into the sterile specimen jars.Ten water trough samples were collected in duplicate (n = 20) at different troughs for each barn type (Figure 1).Ten barn aisle samples were collected in triplicate (n = 30) from sites throughout the barn for each barn type.Ten sump pond samples were collected in duplicate (n = 20) from numerous sites around each of the two ponds that corresponded to each barn ventilation type.In total, 70 samples were collected representing each barn ventilation type for a total of 140 samples.The samples were stored on ice and transported back to the United States Department of Agriculture (USDA) laboratory in College Station, Texas.Upon return to the lab, 50 mL of alkaline peptone water enrichment broth was added to each jar and the jars were incubated overnight at 37 °C.

Aeromonas Isolation and Identification
For isolation, Aeromonas medium Base (RYAN) (Oxoid TM ThermoFisher Scientific, Waltham, MA, United States) with Oxoid TM Ampicillin Selective Supplement (ThermoFisher Scientific cat.no.SR0136) at 5.0 mg/500 mL was prepared according to the manufacturer's instructions and EPA Method 1605 [37,38].Ten milliliters of enrichment broth were filtered through a 2.2 µm 60 mm filter.The filters were placed right-side up on the m-Aeromonas selection agar plates and incubated overnight at 35 °C.Aeromonas was presumptively identified by the production of acid from dextrin fermentation and bright yellow colonies that were greater than 0.5 mm.Additional microbiological testing was

Management area with cross ventilation system
Management area with flow-through ventilation system used to confirm the genus and speciate of the isolates.This included: catalase, oxidase, indole testing, Voges-Proskauer (VP), and hemolysis on blood agar.Biolog (Biolog, Hayward, CA, USA) phenotyping was performed using a GN2 MicroPlate TM .

Statistical Testing
Pearson's chi-squared test was used to determine statistical significance for Aeromonas isolation associated with the flow-through and cross-vent barn locations.

Phylogenetic Tree Construction
A phylogenetic tree was constructed using the Phylogenetic Tree Building Service on PATRIC.This service, "uses the amino acid and nucleotide sequences from defined number of the BV-BRC global Protein Families … which are picked randomly, to build an alignment, and then generate a tree based on the differences within those (randomly) selected sequences (chosen by PATRIC software)."[48].In addition to the experimental isolates (n = 22), genomes were selected from the PATRIC database for 10 different species of Aeromonas.If available, at least five genomes from the ten species were selected.When less than five genomes were available, all were selected.If there were more than five, the choices were based on location and date.Additionally, the reference genome from the National Center for Biotechnology Information (NCBI) was downloaded for each of the 10 Aeromonas species.A total of 53 additional genomes were selected: A. hydrophila (n = 7), including A. hydrophila ATCC 7966 and A. hydrophila CVM861, A. media (n = 6), A. dhakensis (n = 6), A. enteropelogenes (n = 6), A. jandaei (n = 5), A. veronii (n = 6), A. bestiarum (n = 3), A. eucrenophila (n = 2), A. salmonicida (n = 6), and A. caviae (n = 6).The phylogenetic tree was displayed using the Interactive Tree of Life (https://itol.embl.de/,accessed on) [49].

WGS Phylogeny
WGS were submitted to NCBI as raw reads.The SRA and biosample accession numbers are in Table 1.The BioProject study number was PRJNA72334.The phylogenetic tree suggested the Aeromonas spp.were also comprised of four species, A. caviae (n = 15), A. hydrophila (n = 4), A. salmonicida (n = 2), and A. dhakensis (n = 1), but the results were different from the Biolog results (Table 1, Figure 3).WGS isolates A2, A5, A6, A9, A13, and A18 were likely the same clone that grouped together on the dendrogram (Figure 3).Overall, all the 15 A. caviae from the samples were close to the selected A. caviae on the tree.

Discussion
This study reports four Aeromonas spp. on a dairy farm of 1000 healthy Holstein cattle in Texas.Aeromonas clones were disseminated across the physical area of the working farm where the samples were collected.
WGS and phylogenetic analysis of the Aeromonas genomes separated taxonomic clusters into four species, A. hydrophila, A. caviae, A. salmonicida, and A. dhakensis, while the Biolog system identified slightly different results based on biochemical profiles.The Biolog profiles did not include A. dhakensis.This illustrates the importance of Whole Genome Sequencing to confirm biochemical identifications.
A. caviae A14, A15, and A16 were isolated from the cross-vent-associated sump pond and are likely clones with genetic differences only in mobility elements.Evaluation of all genetic traits indicated A2, A5, A6, A9, A13, and A18 were A. caviae clones.They all possessed the blaMOX-6 and blaMOX-7 beta-lactamase genes, the mobility element ISAeme21, and virulence genes sycX and tppE. A. caviae A2, A3, A6, and A18 were genotypically identical and differed from A9 and A13 by the possession of ISAs19 in the latter two.This clone was distributed across different areas of the dairy farm where sample collection was allowed (Figure 2).This illustrates that the type of ventilation management was not a factor in bacterial distribution and suggests that Aeromonas spp.are a ubiquitous environmental resident that could be found anywhere on a farm this large regardless of these two management types.
In addition to elucidating phylogenetic relationships among the farm isolates, genomic features could be compared to A. hydrophila CVM861 and other Aeromonas isolates that have been sequenced.WGS of the 22 dairy isolates gives insight into a pool of mobile elements (e.g., plasmids, transposons, integrons, and Integrative Conjugative Elements (ICEs), Miniature Inverted-Repeat Transposable Elements (MITEs), and (IS) insertion sequences) that may lead to the dissemination of virulence and antimicrobial resistance genes.The first published whole genome sequence of an Aeromonas strain was A. hydrophila ATCC 7966.This strain had no transposase or insertion sequences, which was considered unusual for a 47 Mb genome [50].In our study, A. hydrophila A3, A. dhakensis A10, and A. caviae A17 and A20 had no mobility elements.A. hydrophila A3 and A. dhakensis A10 possessed the same two resistance genes, ampH_1 and ampH_2.However, A3 was phenotypically pansusceptible, suggesting the beta-lactamases were not expressed or capable of conferring resistance. A. caviae A4 and A12 each had the same two transposons: Tn6180 was initially detected in Acinetobacter baumannii in Singapore [51] and Tn6279 was initially detected in Acinetobacter baumannii in Sweden [52]. A. hydrophila CVM861 harbors plasmid IncQ1_1_M28829, which likely carries the Tn21 transposon that is adjacent to the class I integron and macrolide resistance gene operon.In comparison, none of the dairy isolates had alignments to any plasmids.In this report, genotypic analysis showed that all A. caviae harbored one or two β-lactamase genes above the 95% identity cut-off.A. caviae A12 possessed only one β-lactamase gene, but also had a tet(E) gene.The A. hydrophila isolates had one to three β-lactamase genes above the 95% identity cut-off.A. salmonicida A19 had the cphA5 gene and A. salmonicida A23 had the cphA1 gene.
Phenotypically, all A. caviae were resistant to ampicillin.In addition, A. caviae A12 was also resistant to tetracycline.This was consistent with the genotypic analysis.The A. hydrophila isolates were all resistant to one or more β-lactams, which was also consistent with the analysis.A. salmonicida A23 was resistant to ampicillin and also had a β-lactamase gene.A. salmonicida A19, however, was phenotypically pansusceptible despite having a β-lactamase gene.
Aeromonas spp.secrete a wide variety of extracellular enzymes that are considered virulence factors involved in pathogenesis.These genes encode for proteins that include hemolysins, aerolysins, enterotoxins, cytotoxins, and others.Aeromonas spp.can regulate gene expression by quorum sensing, which is also coordinated with biofilm formation [15,53].Several of the A. caviae, A2, A5, A6, A7, A8, A9, A13, and A18, possess the exeE gene, necessary for extracellular export and normal outer membrane assembly [54].A previous study identified Aeromonas isolates from cattle that exhibited resistance to carbapenem [55].This study did not screen for resistance to carbapenem, but several relevant genes (cphA1_4_AY261376, cphA5_4_AY22051, and cphA1_AY261379) were identified.
The different ventilation systems did not appear to influence the recovery of Aeromonas isolates.Even though there were different ventilation systems present the cattle were in relatively close proximity to each other.Water runoff, biting flies, and a number of environmental factors could have easily transmitted bacteria between the herds.Although we did encounter some antibiotic resistance among the isolates, none displayed multidrug resistance.The mobility elements and virulence genes suggested they were localized to the chromosome and were unlikely to be horizontally transferred to other bacterial genera.Thus, on a dairy farm of 1000 healthy cattle, the existence of these 22 Aeromonas isolates could be considered as normal environmental flora.

Figure 1 .
Figure 1.Location of samples on the farm.

Figure 2 .
Figure 2. Location of Aeromonas positives on the farm.

Table 1 .
Aeromonas spp.collected from a central Texas dairy farm.
(n represents total samples in each area) Management area with flow-through ventilation system *, **, *** Isolates with same number of asterisks were most closely related (average nucleotide identity of >99.5% with each other) Isolates without asterisks were less related

Table 2 .
Percent alignment of genes identified in Aeromonas hydrophila, Aeromonas salmonicida, and Aeromonas caviae isolates from a central Texas dairy farm to antimicrobial resistance genes.

Table 3 .
Percent alignment of genes identified in Aeromonas hydrophila, Aeromonas salmonicida, and Aeromonas caviae isolates from a central Texas dairy farm to genes in the MobileElementFinder database.

Table 4 .
Percent alignment of genes identified in Aeromonas hydrophila, Aeromonas salmonicida, and Aeromonas caviae isolates from a central Texas dairy farm to genes in the Virulence Factor database.

Table 5 .
Percent alignment of genes identified in Aeromonas hydrophila isolates from a central Texas dairy farm to Aeromonas-specific virulence genes selected from GenBank.