Shifts in bacterial communities and antibiotic resistance genes in surface water and gut microbiota of guppies (Poecilia reticulata) in the upper Rio Uberabinha, Brazil.

Anthropogenic activities especially water pollution can affect the diversity and composition of microbial communities and promote the spread of antibiotic resistance genes (ARGs). In this study, water samples and guppies (Poecilia reticulata) were sampled from six sampling sites along the Uberabinha River in southeastern Brazil, both microbial communities and ARGs of surface waters and intestinal microbiota of guppies (Poecilia reticulata) were detected. According to the results of 16S rRNA amplicon sequencing, Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria were dominant phyla in both water and intestinal microbiota, but the abundance of putative pathogens was higher at heavily polluted sites. Up to 83% of bacteria in intestinal microbiota originated from water microbiota; this proportion was relatively higher in less polluted compared to polluted environments. ARGs providing resistance of tetracyclines and quinolones were dominant in both water and gut microbiota. The relative abundances of class I integrons and ARGs were as high as 1.74 × 10-1/16S rRNA copies and 3.61 × 10-1/16S rRNA copies, respectively, at heavily polluted sites. Correlation analysis suggests that integrons and bacteria play key roles in explaining the widespread occurrence of ARGs in the surface, but not in intestinal microbiota. We could rule out the class I integrons a potential intermediary bridge for ARGs between both types of microbiomes. Our results highlight the tight link in microbial communities and ARGs between ambient microbiota of stream ecosystems and intestinal microbiota of fish. Our study could have far-reaching consequences for fisheries and consumer safety and calls for investigations of gut microbiota of target species of both commercial fisheries and recreational (hobby) angling.

Phylogenetic characteristics, virulence properties and antibiogram profile of motile Aeromonas spp. isolated from ornamental guppy (Poecilia reticulata).

Aeromonas spp. are opportunistic pathogenic bacteria related to an assembly of infectious diseases in ornamental fish. In the present study, virulence properties and antibiotic susceptibility of 52 guppy-borne Aeromonas spp. were investigated. The isolates were identified as A. veronii (n = 34), A. dhakensis (n = 10), A. hydrophila (n = 3), A. caviae (n = 3) and A. enteropelogenes (n = 2) by gyrB gene sequencing. The gyrB sequence deviation within and among the species ranged from 0 to 2.6% and 2.7-9.2%. Each species formed a distinct group in the unrooted neighbor-joining phylogenetic tree. The phenotypic virulence factors such as ß-hemolysis, slime, caseinase, DNase, gelatinase and lipase production were observed in 28 (53.9%), 33 (63.5%), 28 (53.9%), 42 (80.8%), 37 (71.2%) and 42 (80.8%) isolates, respectively. The virulence genes were detected by PCR assay in the following proportions- act (84.6%), hly (80.8%), aer (73.1%), lip (73.1%), gcaT (73.1%), ascV (53.8%), ahyB (53.8%) fla (51.9%), alt (48.1%), ast (36.5%) and ser (34.6%), respectively. The amoxicillin, ampicillin, imipenem, nalidixic acid, oxytetracycline and rifampicin were resistant to more than 70.0% of the isolates in antibiotic susceptibility test. Our study suggests that the ornamental guppy can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp.

Incidence of antimicrobial resistance genes and class 1 integron gene cassettes in multidrug-resistant motile Aeromonas sp. isolated from ornamental guppy (Poecilia reticulata).

Aeromonas sp. are opportunistic pathogenic bacteria which are associated with various diseases in ornamental fish, aquaculture raised species and wild fisheries. In our study, antimicrobial resistance patterns, antimicrobial resistance genes and class 1 integron gene cassettes of 52 guppy-borne Aeromonas sp. were examined. The isolates were identified as A. veronii (n = 34), A. dhakensis (n = 10), A. hydrophila (n = 3), A. caviae (n = 3) and A. enteropelogenes (n = 2) by gyrB gene sequencing. Every isolate was resistant to at least four antimicrobials in disc diffusion test. The resistance to amoxicillin, nalidixic acid and oxytetracycline was 100% among the tested isolates. 92·30, 76·92, 71·15, 51·92, 51·92 and 50·00% of the isolates were resistant to ampicillin, rifampicin, imipenem, cephalothin, tetracycline and trimethoprim respectively. The multiple antibiotic resistance index values ranged from 0·28 to 0·67. PCR amplification of antimicrobial resistance genes implied the occurrence of tetracycline resistance (tetA (65·39%), tetE (25·00%) and tetB (15·38%)), plasmid-mediated quinolone resistance (qnrS (26·92%) and qnrB (17·31%)) and aminoglycoside resistance (aphaAI-IAB (7·69%) and aac (6')-Ib (3·84%)) genes in the isolates. The IntI gene was positive for 36·54% of the isolates and four class 1 integron gene cassette profiles (aadA2, qacE2-orfD, aadA2-catB2 and dfrA12-aadA2) were identified. These data suggest that ornamental guppy can be a reservoir of multidrug-resistant Aeromonas sp. which comprise different antimicrobial resistance genes and class 1 integrons. SIGNIFICANCE AND IMPACT OF THE STUDY: Antimicrobial resistance genes and integron gene cassettes of ornamental fish-borne aeromonads are poorly studied. The antimicrobial resistance patterns, antimicrobial resistance genes and class 1 integron gene cassettes of Aeromonas sp. isolated from ornamental guppy were characterized for the first time in Korea. The incidence of different antimicrobial resistance genes and class 1 integron gene cassettes were observed in multidrug-resistant Aeromonas isolates. This result suggests that better management practices are necessary to prevent and address the serious consequences of indiscriminate and inappropriate antimicrobial use, and the distribution of multidrug-resistant bacteria.

Evaluation of the Impacts of Long-Term Enriched Artemia with Bacillus subtilis on Growth Performance, Reproduction, Intestinal Microflora, and Resistance to Aeromonas hydrophila of Ornamental Fish Poecilia latipinna.

The present study investigated the effect of enriched Artemia with Bacillus subtilis on growth performance, reproductive factors, proximate composition, intestinal microflora, and resistance to Aeromonas hydrophila of ornamental fish, Poecilia latipinna. Using a completely randomized design, the experiment included three groups. The first group was fed with commercial food without any probiotic. The second group was fed with unenriched Artemia, and the last group consumed long-time enriched Artemia with Bacillus subtilis. The bacteria B. subtilis with a density of 1 × 105 CFU mL-1 was added daily to Artemia culture medium. The total microflora and Bacillus subtilis counts were significantly increased in enriched Artemia compared to the unenriched group (P < 0.05). In fish fed groups, growth factors did not show any significant difference (P > 0.05). The maximum relative fecundity (28.65 ± 2.52 egg number g-1), fry production (62.93 ± 4.6 individual per female), and fry survival (70.97 ± 1.56%) obtained in the third group were found to be significantly more than those in the first and the second groups. Moreover, intestinal bacterial count for Bacillus revealed that the higher concentration of bacteria was significantly related to the third group (6.24 ± 0.11 log CFU g-1) (P < 0.05). Maximum protein and fat contents were observed in fish fed with Bacillus-enriched Artemia; however, no significant difference was found between control and unenriched Artemia groups (P > 0.05). The highest amount of ash was observed in fish fed with commercial food without any probiotic (P < 0.05). At the end of the feeding period, each of the three groups along with positive group (oxytetracycline 100 mg kg-1 of commercial food) was exposed to A. hydrophila (BCCM5/LMG3770) bacteria intraperitoneally. Based on the results, the lowest cumulative mortality was significantly found in group three (68.75 ± 3.6%) and positive group (62.5 ± 7.0%) compared to control and unenriched Artemia groups (P < 0.05). Hence, B. subtilis with a concentration of 1 × 105 CFU mL-1 during the period of Artemia culturing can improve the reproductive parameters, intestinal microflora, and resistance to pathogenic bacteria of Poecilia latipinna.

Pathogenic characteristics of Aeromonas veronii isolated from the liver of a diseased guppy (Poecilia reticulata).

Despite the significant development on their diagnoses and control, aeromonad infection is still a problem in aquaculture. This study described the key bacteriological and pathogenic features of a presumptive Aeromonas sp. isolated from the liver of a diseased guppy (Poecilia reticulata). Molecular identification revealed that the isolate was an Aeromonas veronii (A. veronii PR). It was able to grow in a wide range of temperatures and salt concentrations, and was capable of auto-aggregation and biofilm formation, with temperature as an influencing factor. Some of the extracellular enzymes that may be involved in its virulence include caseinase, gelatinase and lipase. The infection rate was relatively progressive, and fish with prior infection showed marginal resistance to secondary infection. Handling stress differentially influenced the infection kinetics at the early stages; however, the final mortality rates did not significantly differ between the groups. A comparative infection trial revealed that zebrafish (Danio rerio) were more susceptible to A. veronii PR than guppy. The presented intrinsic and extrinsic factors influencing the pathogenicity of A. veronii PR lay the foundation for future research to better understand this pathogen in freshwater ornamental fish aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonad infections continue to affect the fish farming industry. Several new species of Aeromonads in freshwater ornamental fish have been identified in the last years. In this study, we have characterized an Aeromonas veronii isolate from a diseased guppy. The series of experiments identified the intrinsic and extrinsic factors contributing to the pathogenic characteristics of the isolate. It has been shown to be pathogenic to both guppy and zebrafish. The results offer foundational knowledge in the development of preventive and therapeutic measures to combat this pathogen in the ornamental fish industry.

Emerging human pathogen Acinetobacter baumannii in the natural aquatic environment: a public health risk?

Bacterium Acinetobacter baumannii is an emerging human pathogen whose presence in the aquatic environment raises the issue of public health risk. Fish colonization represents the potential route of pathogen transmission to humans. The aim was to examine the colonization of A. baumannii to freshwater fish Poecilia reticulata. An extensively drug-resistant A. baumannii was tested at three concentrations in natural spring water. Additionally, 70 fish from the Sava River (Croatia) were screened for the presence of A. baumannii, which was not found in gill swabs or analysed gut. The colonization potential of A. baumannii in freshwater fish is dependent upon its concentration in surrounding water. The low concentration of A. baumannii in natural waters represents low colonization potential of freshwater fish. The risk for public health exists in closed water bodies where there is constant inflow of water polluted by A. baumannii in concentrations above 3 log CFU mL-1.

Non-tuberculous Mycobacterium species causing mycobacteriosis in farmed aquatic animals of South Africa.

BACKGROUND: Mycobacteriosis caused by non-tuberculous mycobacteria (NTM), is among the most chronic diseases of aquatic animals. In addition, fish mycobacteriosis has substantial economic consequences especially in the aquaculture and fisheries industry as infections may significantly decrease production and trade. Some fish NTM pathogens are highly virulent and zoonotic; as such, infection of aquaria with these pathogens is a public health concern. In this study, we report isolation of nine different NTM species from sixteen aquatic animals including different fish species, frogs and a crocodile. Given the clinical significance of Mycobacterium marinum and its close relation to Mycobacterium tuberculosis, as well as the significance of ESAT 6 and CFP-10 secretion in mycobacterial virulence, we analysed the esxA and esxB nucleotide sequences of M. marinum isolates identified in this study as well as other mycobacteria in the public databases. RESULTS: Mycobacterium shimoidei, Mycobacterium marinum, Mycobacterium chelonae, Mycobacterium septicum /M. peregrinum and Mycobacterium porcinum were isolated from gold fish, Guppy, exotic fish species in South Africa, koi and undefined fish, Knysna seahorse, as well Natal ghost frogs respectively, presenting tuberculosis like granuloma. Other NTM species were isolated from the studied aquatic animals without any visible lesions, and these include Mycobacterium sp. N845 T, Mycobacterium fortuitum, a member of the Mycobacterium avium complex, and Mycobacterium szulgai. Phylogenetic analysis of mycobacteria, based on esxA and esxB genes, separated slow growing from rapidly growing mycobacteria as well as pathogenic from non-pathogenic mycobacteria in some cases. CONCLUSIONS: Isolation of the different NTM species from samples presenting granuloma suggests the significance of these NTM species in causing mycobacteriosis in these aquatic animals. The study also revealed the potential of esxA and esxB sequences as markers for phylogenetic classification of mycobacteria. Observations regarding use of esxA and esxB sequences for prediction of potential pathogenicity of mycobacteria warrants further investigation of these two genes in a study employing NTM species with well-defined pathogenicity.

Community assembly of a euryhaline fish microbiome during salinity acclimation.

Microbiomes play a critical role in promoting a range of host functions. Microbiome function, in turn, is dependent on its community composition. Yet, how microbiome taxa are assembled from their regional species pool remains unclear. Many possible drivers have been hypothesized, including deterministic processes of competition, stochastic processes of colonization and migration, and physiological 'host-effect' habitat filters. The contribution of each to assembly in nascent or perturbed microbiomes is important for understanding host-microbe interactions and host health. In this study, we characterized the bacterial communities in a euryhaline fish and the surrounding tank water during salinity acclimation. To assess the relative influence of stochastic versus deterministic processes in fish microbiome assembly, we manipulated the bacterial species pool around each fish by changing the salinity of aquarium water. Our results show a complete and repeatable turnover of dominant bacterial taxa in the microbiomes from individuals of the same species after acclimation to the same salinity. We show that changes in fish microbiomes are not correlated with corresponding changes to abundant taxa in tank water communities and that the dominant taxa in fish microbiomes are rare in the aquatic surroundings, and vice versa. Our results suggest that bacterial taxa best able to compete within the unique host environment at a given salinity appropriate the most niche space, independent of their relative abundance in tank water communities. In this experiment, deterministic processes appear to drive fish microbiome assembly, with little evidence for stochastic colonization.

Divergence across diet, time and populations rules out parallel evolution in the gut microbiomes of Trinidadian guppies.

Diverse microbial consortia profoundly influence animal biology, necessitating an understanding of microbiome variation in studies of animal adaptation. Yet, little is known about such variability among fish, in spite of their importance in aquatic ecosystems. The Trinidadian guppy, Poecilia reticulata, is an intriguing candidate to test microbiome-related hypotheses on the drivers and consequences of animal adaptation, given the recent parallel origins of a similar ecotype across streams. To assess the relationships between the microbiome and host adaptation, we used 16S rRNA amplicon sequencing to characterize gut bacteria of two guppy ecotypes with known divergence in diet, life history, physiology and morphology collected from low-predation (LP) and high-predation (HP) habitats in four Trinidadian streams. Guts were populated by several recurring, core bacteria that are related to other fish associates and rarely detected in the environment. Although gut communities of lab-reared guppies differed from those in the wild, microbiome divergence between ecotypes from the same stream was evident under identical rearing conditions, suggesting host genetic divergence can affect associations with gut bacteria. In the field, gut communities varied over time, across streams and between ecotypes in a stream-specific manner. This latter finding, along with PICRUSt predictions of metagenome function, argues against strong parallelism of the gut microbiome in association with LP ecotype evolution. Thus, bacteria cannot be invoked in facilitating the heightened reliance of LP guppies on lower-quality diets. We argue that the macroevolutionary microbiome convergence seen across animals with similar diets may be a signature of secondary microbial shifts arising some time after host-driven adaptation.

Parasites pitched against nature: Pitch Lake water protects guppies (Poecilia reticulata) from microbial and gyrodactylid infections.

SUMMARY The enemy release hypothesis proposes that in parasite depleted habitats, populations will experience relaxed selection and become more susceptible (or less tolerant) to pathogenic infections. Here, we focus on a population of guppies (Poecilia reticulata) that are found in an extreme environment (the Pitch Lake, Trinidad) and examine whether this habitat represents a refuge from parasites. We investigated the efficacy of pitch in preventing microbial infections in Pitch Lake guppies, by exposing them to dechlorinated water, and reducing gyrodactylid infections on non-Pitch Lake guppies by transferring them to Pitch Lake water. We show that (i) natural prevalence of ectoparasites in the Pitch Lake is low compared to reference populations, (ii) Pitch Lake guppies transferred into aquarium water develop microbial infections, and (iii) experimentally infected guppies are cured of their gyrodactylid infections both by natural Pitch Lake water and by dechlorinated water containing solid pitch. These results indicate a role for Pitch Lake water in the defence of guppies from their parasites and suggest that Pitch Lake guppies might have undergone enemy release in this extreme environment. The Pitch Lake provides an ideal ecosystem for studies on immune gene evolution in the absence of parasites and long-term evolutionary implications of hydrocarbon pollution for vertebrates.

Speciation in caves: experimental evidence that permanent darkness promotes reproductive isolation.

Divergent selection through biotic factors like predation or parasitism can promote reproductive isolation even in the absence of geographical barriers. On the other hand, evidence for a role of adaptation to abiotic factors during ecological speciation in animals is scant. In particular, the role played by perpetual darkness in establishing reproductive isolation in cave animals (troglobites) remains elusive. We focused on two reproductively isolated ecotypes (surface- and cave-dwelling) of the widespread livebearer Poecilia mexicana, and raised offspring of wild-caught females to sexual maturity in a 12-month common-garden experiment. Fish were reared in light or darkness combined with high- or low-food conditions. Females, but not males, of the surface ecotype suffered from almost complete reproductive failure in darkness, especially in the low-food treatment. Furthermore, surface fish suffered from a significantly higher rate of spontaneous, stress-related infection with bacterial columnaris disease. This experimental evidence for strong selection by permanent darkness on non-adapted surface-dwelling animals adds depth to our understanding of the selective forces establishing and maintaining reproductive isolation in cave faunas.