Unveiling novel threats: Urban river isolation of Aeromonas veronii with unusual VEB-28 extended-spectrum ß-lactamase and distinct mcr variants.

Aeromonas spp. are frequently encountered in aquatic environments, with Aeromonas veronii emerging as an opportunistic pathogen causing a range of diseases in both humans and animals. Recent reports have raised public health concerns due to the emergence of multidrug-resistant Aeromonas spp. This is particularly noteworthy as these species have demonstrated the ability to acquire and transmit antimicrobial resistance genes (ARGs). In this study, we report the genomic and phenotypic characteristics of the A. veronii TR112 strain, which harbors a novel variant of the Vietnamese Extended-spectrum ß-lactamase-encoding gene, blaVEB-28, and two mcr variants recovered from an urban river located in the Metropolitan Region of São Paulo, Brazil. A. veronii TR112 strain exhibited high minimum inhibitory concentrations (MICs) for ceftazidime (64 µg/mL), polymyxin (8 µg/mL), and ciprofloxacin (64 µg/mL). Furthermore, the TR112 strain demonstrated adherence to HeLa and Caco-2 cells within 3 h, cytotoxicity to HeLa cells after 24 h of interaction, and high mortality rates to the Galleria mellonella model. Genomic analysis showed that the TR112 strain belongs to ST257 and presented a range of ARGs conferring resistance to ß-lactams (blaVEB-28, blaCphA3, blaOXA-912) and polymyxins (mcr-3 and mcr-3.6). Additionally, we identified a diversity of virulence factor-encoding genes, including those encoding mannose-sensitive hemagglutinin (Msh) pilus, polar flagella, type IV pili, type II secretion system (T2SS), aerolysin (AerA), cytotoxic enterotoxin (Act), hemolysin (HlyA), hemolysin III (HlyIII), thermostable hemolysin (TH), and capsular polysaccharide (CPS). In conclusion, our findings suggest that A. veronii may serve as an environmental reservoir for ARGs and virulence factors, highlighting its importance as a potential pathogen in public health.

Structural Studies of the Lipopolysaccharide of Aeromonas veronii bv. sobria Strain K133 Which Represents New Provisional Serogroup PGO1 Prevailing among Mesophilic Aeromonads on Polish Fish Farms.

In the present work, we performed immunochemical studies of LPS, especially the O-specific polysaccharide (O-PS) of Aeromonas veronii bv. sobria strain K133, which was isolated from the kidney of carp (Cyprinus carpio L.) during an outbreak of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) on a Polish fish farm. The structural characterization of the O-PS, which was obtained by mild acid degradation of the LPS, was performed with chemical methods, MALDI-TOF mass spectrometry, and 1H and 13C NMR spectroscopy. It was revealed that the O-PS has a unique composition of a linear tetrasaccharide repeating unit and contains a rarely occurring sugar 2,4-diamino-2,4,6-trideoxy-D-glucose (bacillosamine), which may determine the specificity of the serogroup. Western blotting and ELISA confirmed that A. veronii bv. sobria strain K133 belongs to the new serogroup PGO1, which is one of the most commonly represented immunotypes among carp and trout isolates of Aeromonas sp. in Polish aquacultures. Considering the increase in the MAI/MAS incidences and their impact on freshwater species, also with economic importance, and in the absence of an effective immunoprophylaxis, studies of the Aeromonas O-antigens are relevant in the light of epidemiological data and monitoring emergent pathogens representing unknown antigenic variants and serotypes.

Genome sequencing and annotation of multi-virulent Aeromonas veronii XhG1.2 isolated from diseased Xiphophorus hellerii.

The present study was intended to elucidate the genomic basis of antibiotic resistance and hyper-virulence of the fish pathogen Aeromonas veronii XhG1.2 characterized in our previous work. The identity of XhG1.2 was confirmed through 16S rDNA sequence analysis and whole genome sequence analysis. The top-hit species distribution analysis of XhG1.2 sequence data revealed major hits against the Aeromonas veronii. The identification of virulence genes using the VFDB showed the genome of XhG1.2 to have the genes coding for the virulence factors viz. aerolysin, RtxA, T2SS, T3SS and T6SS. The presence of antibiotic resistance predicted through the CARD database analysis showed it to have the CephA3, OXA-12, adeF and pulvomycin resistance genes. By the phylogenetic and comparative genomic analysis, A. veronii species were found to have genes for toxin production. This also confirmed the pathogenicity and drug resistance of A. veronii XhG1.2 and also its potential to cause disease in diverse ornamental fishes.

Urinary tract infection due to Aeromonas species: An uncommon causative agent.

BACKGROUND: Members of the genus Aeromonas are Gram-negative bacilli, belonging to family Aeromonadaceae, and are widely found in various aquatic environments. The most common species associated with human infections are A. hydrophila, A. caviae, and A. veronii biovar sobria. Aeromonas species are recognized as emerging opportunistic pathogens in humans mainly causing gastrointestinal infections and wound infections with or without progression to septicaemia. Aeromonas organisms rarely cause urinary tract infection (UTI) and are not known uropathogens. CASE: We report a series of UTI due to Aeromonas species in three adult patients, specifically identified as A. veronii biovar sobria in two patients and A. hydrophila in one patient. Two patients had history of occupational exposure to aquatic environment. CONCLUSIONS: The cases highlight another expanded range of infections caused by Aeromonas spp. that can be encountered in a community setting and indicate that infections with Aeromonas spp. should be kept in mind while investigating for the etiology of UTI, especially in adult patients with occupational exposure to aquatic ecosystems.

Cadaverine reverse transporter (CadB protein) contributes to the virulence of Aeromonas veronii TH0426.

Aeromonas veronii is one of the main pathogens causing sepsis and ulcer syndrome in freshwater fish. Analysis of the results of epidemiological investigations in recent years has revealed that the virulence of A. veronii and its tolerance to drugs have been increasing year by year. Currently, most of the research on A. veronii focuses on its isolation, identification, and drug susceptibility, whereas research on its virulence factors and pathogenesis mechanisms is relatively rare. In this study, we identified and obtained the highly expressed TH0426 cadaverine reverse transporter (CadB) of A. veronii. We used efficient suicide plasmid-mediated homologous recombination to delete the cadB gene in TH0426 and constructed a cadB deletion strain. The LD50 of ΔcadB was 93.2 times higher than that of TH0426 in zebrafish, the toxicity of ΔcadB was 9.5 times less than that of TH0426 in EPC cells, and the biofilm formation ability of ΔcadB was 5.6-fold greater than that of TH0426. In addition, motility detection results indicated that ΔcadB had lost its swimming ability. The results of flagellar staining and TEM demonstrated that ΔcadB shed the flagella. In summary, the virulence and adhesion of A. veronii TH0426 were significantly decreased by the deletion of cadB, which might provide a theoretical basis for research into A. veronii virulence factors.

Highly accurate-single chromosomal complete genomes using IonTorrent and MinION sequencing of clinical pathogens.

Oxford Nanopore MinION sequencing technology has been gaining immense importance in identification of pathogen and antimicrobial resistance, though with 10-15% error rate. Short read technologies generates high accurate genome but with multiple fragments of genome. This study proposes a novel workflow to reduce the indels resulted from MinION long read sequencing by overlaying short read sequences from IonTorrent in the clinical isolates. Best of both techniques were employed which generated highly accurate-single chromosomal microbial genomes with increase in completeness of genomes from 44.5%, 30% and 43% to 98.6%, 98.6% and 96.6% for P. aeruginosa, A. veronii and B. pertussis respectively. To the best of our knowledge, this is the first study to generate a hybrid of IonTorrent and MinION reads to obtain single chromosomal genomes. This would enable to precisely infer both structural arrangement of genes and SNP based analysis for phylogenetic information.

Prevalence of Potentially Pathogenic Antibiotic-Resistant Aeromonas spp. in Treated Urban Wastewater Effluents versus Recipient Riverine Populations: a 3-Year Comparative Study.

Antibiotic resistance continues to be an emerging threat both in clinical and environmental settings. Among the many causes, the impact of postchlorinated human wastewater on antibiotic resistance has not been well studied. Our study compared antibiotic susceptibility among Aeromonas spp. in postchlorinated effluents to that of the recipient riverine populations for three consecutive years against 12 antibiotics. Aeromonas veronii and Aeromonas hydrophila predominated among both aquatic environments, although greater species diversity was evident in treated wastewater. Overall, treated wastewater contained a higher prevalence of nalidixic acid-, trimethoprim-sulfamethoxazole (SXT)-, and tetracycline-resistant isolates, as well as multidrug-resistant (MDR) isolates compared to upstream surface water. After selecting for tetracycline-resistant strains, 34.8% of wastewater isolates compared to 8.3% of surface water isolates were multidrug resistant, with nalidixic acid, streptomycin, and SXT being the most common. Among tetracycline-resistant isolates, efflux pump genes tetE and tetA were the most prevalent, though stronger resistance correlated with tetA. Over 50% of river and treated wastewater isolates exhibited cytotoxicity that was significantly correlated with serine protease activity, suggesting many MDR strains from effluent have the potential to be pathogenic. These findings highlight that conventionally treated wastewater remains a reservoir of resistant, potentially pathogenic bacterial populations being introduced into aquatic systems that could pose a threat to both the environment and public health.IMPORTANCE Aeromonads are Gram-negative, asporogenous rod-shaped bacteria that are autochthonous in fresh and brackish waters. Their pathogenic nature in poikilotherms and mammals, including humans, pose serious environmental and public health concerns especially with rising levels of antibiotic resistance. Wastewater treatment facilities serve as major reservoirs for the dissemination of antibiotic resistance genes (ARGs) and resistant bacterial populations and are, thus, a potential major contributor to resistant populations in aquatic ecosystems. However, few longitudinal studies exist analyzing resistance among human wastewater effluents and their recipient aquatic environments. In this study, considering their ubiquitous nature in aquatic environments, we used Aeromonas spp. as bacterial indicators of environmental antimicrobial resistance, comparing it to that in postchlorinated wastewater effluents over 3 years. Furthermore, we assessed the potential of these resistant populations to be pathogenic, thus elaborating on their potential public health threat.

Comparative genomics of Aeromonas veronii: Identification of a pathotype impacting aquaculture globally.

Aeromonas veronii is a gram-negative species abundant in aquatic environments that causes disease in humans as well as terrestrial and aquatic animals. In the current study, 41 publicly available A. veronii genomes were compared to investigate distribution of putative virulence genes, global dissemination of pathotypes, and potential mechanisms of virulence. The complete genome of A. veronii strain ML09-123 from an outbreak of motile aeromonas septicemia in farm-raised catfish in the southeastern United States was included. Dissemination of A. veronii strain types was discovered in dispersed geographical locations. Isolate ML09-123 is highly similar to Chinese isolate TH0426, suggesting the two strains have a common origin and may represent a pathotype impacting aquaculture in both countries. Virulence of strain ML09-123 in catfish in a dose-dependent manner was confirmed experimentally. Subsystem category disposition showed the majority of genomes exhibit similar distribution of genomic elements. The type I secretion system (T1SS), type II secretion system (T2SS), type 4 pilus (T4P), and flagellum core elements are conserved in all A. veronii genomes, whereas the type III secretion system (T3SS), type V secretion system (T5SS), type VI secretion system (T6SS), and tight adherence (TAD) system demonstrate variable dispersal. Distribution of mobile elements is dependent on host and geographic origin, suggesting this species has undergone considerable genetic exchange. The data presented here lends insight into the genomic variation of A. veronii and identifies a pathotype impacting aquaculture globally.

Characterization of virulence properties and multi-drug resistance profiles in motile Aeromonas spp. isolated from zebrafish (Danio rerio).

Aeromonas spp. are opportunistic pathogenic bacteria associated with a multitude of diseases in ornamental fish. In this study, virulence properties and antibiotic resistance patterns of 43 Aeromonas strains isolated from 46 zebrafish were investigated. The isolates were identified as Aeromonas veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), Aeromonas hydrophila (n = 8), A. caviae (n = 3), Aeromonas enteropelogenes (n = 2) and Aeromonas dhakensis (n = 1) by gyrB gene sequencing. The sequence divergence within and between the species ranged from 0-5·80% and 4·90-8·00%. Each species formed a distinct group in a neighbour-joining phylogenetic tree. The lipase production, biofilm formation, DNase activity, gelatinase production, caseinase production and ß-hemolysis were phenotypically observed in 34 (79·07%), 33 (74·74%), 30 (69·77%), 25 (58·14%), 22 (51·18%) and 21 (48·84%) isolates. The virulence genes were detected by polymerase chain reaction (PCR) in following frequencies- aer (86·05%), hlyA (83·72%), gcaT (83·72%), lip (72·09%), act (67·44%), fla (65·12%), ascV (58·14%), ast (55·81%), ser (41·86%), ahyB (39·53%) and alt (25·58%). Every isolate was resistant to at least four antibiotics in disk diffusion test. The multiple antibiotic resistance (MAR) index values ranged from 0·22-0·50 among the isolates. Our study suggests that zebrafish can be a potential reservoir of virulent and multi-drug resistant Aeromonas spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas spp. are Gram-negative and facultative anaerobic bacteria which are ubiquitous in aquatic environments. Virulence properties and antibiotic resistance of ornamental fish-borne Aeromonas spp. are poorly understood. The virulence factors as well as multiple antibiotic resistance profiles of zebrafish-borne Aeromonas spp. were characterized for the first time in Korea. Most of the isolates were positive for phenotypic virulence traits and harboured several virulence genes revealing the virulence potential of zebrafish-borne Aeromonas spp. Additionally, the high multiple antibiotic resistance (MAR) index values displayed by the isolates highlight the necessity of responsible use of antibiotics in the ornamental fish industry.

Biofabrication of silver nanoparticles using bacteria from mangrove swamp.

The last decade has observed a rapid advancement in utilising biological system towards bioremediation of metal ions in the form of respective metal nanostructures or microstructures. The process may also be adopted for respective metal nanoparticle biofabrication. Among different biological methods, bacteria-mediated method is gaining great attention for nanoparticle fabrication due to their eco-friendly and cost-effective process. In the present study, silver nanoparticle (AgNP) was synthesised via continuous biofabrication using Aeromonas veronii, isolated from swamp wetland of Sunderban, West Bengal, India. The biofabricated AgNP was further purified to remove non-conjugated biomolecules using size exclusion chromatography, and the purified AgNPs were characterised using UV-visible spectroscopy, X-ray diffraction, field emission scanning electron microscopy and transmission electron microscopy (TEM). Additionally, the presence of proteins as capping and stabilising agents was confirmed by the amide-I and amide-II peaks in the spectra obtained using attenuated total reflection Fourier transform infrared spectroscopy. The size of biofabricated AgNP was 10-20 nm, as observed using TEM. Additionally, biofabricated AgNP shows significant antibacterial potential against E. coli and S. aureus. Hence, biofabricated AgNP using Aeromonas veronii, which found resistant to a significant concentration of Ag ion, showed enhanced antimicrobial activity compared to commercially available AgNP.

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.

Acute septic arthritis of shoulder joint caused by Aeromonas veronii biotype sobria.

We present the case of a 91-year-old woman who presented with a 2­day history of progressive pain and immobility of the right shoulder joint after fever of unknown etiology. Aeromonas sobria was isolated from a culture of purulent synovial fluid. The clinical condition gradually improved with the application of appropriate antibiotics and no surgical intervention was necessary. This report indicates that acute septic arthritis may result from Aeromonas veronii biotype sobria infections in healthy people. This case is contrary to the previous reports due to the absence of obvious risk factors.

Winter kill in intensively stocked channel catfish (Ictalurus punctatus): Coinfection with Aeromonas veronii, Streptococcus parauberis and Shewanella putrefaciens.

Unusual persistent natural mortality occurred in a floating in-pond raceway system intensively stocked with channel and hybrid catfish beginning in early November 2016 up until March 2017. The temperature during the period of outbreak ranged from 7.2 to 23.7°C. Gross examination of freshly dead and moribund fish revealed pale gills, slight abdominal distension and swollen inflamed vents. Comprehensive necropsy of 20 fish demonstrated vast amounts of bloody ascitic fluid in the coelomic cavity, visceral congestion, splenomegaly and pale friable livers but macroscopically normal kidneys, suggesting systemic bacterial infection. Bacterial cultures were initiated from skin, gills and major internal organs. Following incubation, a mixture of three bacterial colony phenotypes was observed on agar plates. Presumptive biochemical characterization of the isolates followed by 16S-rRNA sequence analysis resulted in the identification of Aeromonas veronii, Streptococcus parauberis and Shewanella putrefaciens. Channel catfish juveniles were experimentally infected with the recovered isolates to fulfil Koch's postulates. Moreover, an antibiogram was used to evaluate the susceptibility of the isolates to antimicrobial drugs approved for use in aquaculture. Aquaflor was used successfully for treatment. Here, we report bacterial coinfection lead by A. veronii and the first identification of S. parauberis and S. putrefaciens from cultured catfish in North America.

Isolation and characterization of a novel, T7-like phage against Aeromonas veronii.

A virulent Aeromonas veronii biovar sobria and the corresponding novel, lytic bacteriophage (VTCCBPA5) were isolated from village pond water. The phage was found to belong to family Podoviridae. PCR analysis of major capsid protein gene confirmed its classification to T7-like genus. The protein profiling by SDS-PAGE indicated the major structural protein to be ~ 45 kDa. The phage (VTCCBPA5) is host specific and is stable over a range of pH (6-10) and temperatures (4-45 °C). On the basis of restriction endonuclease analysis combined with prediction mapping, it was observed to vary significantly from previously reported podophages of Aeromonas sp., viz. phiAS7 and Ahp1. The phylogenetic analysis on the basis of PCR-amplified segment of DNA polymerase gene of phage revealed it being an outgroup from podophages of Klebsiella sp. and Pseudomonas sp. though a small internal fragment (359 bp) showed the highest identity (77%) with Vibrio sp. phages. Thus, this is the first report of a novel Podoviridae phage against A. veronii. It expands the assemblage of podophages against Aeromonas sp. and BPA5 could be potentially useful in biocontrol of environmentally acquired Aeromonas veronii infections.

First isolation and characterization of the pathogenic Aeromonas veronii bv. veronii associated with ulcerative syndrome in the indigenous Pelophylax ridibundus of Al-Ahsaa, Saudi Arabia.

Virulent microbial pathogen infections are the main cause for amphibian decline worldwide. In the present study, a bacterial strain named RDL-2, which was isolated from the skin of infected Pelophylax ridibundus larvae, was cultured and then phenotypically and biochemically characterized using scanning electron microscopic observations and the API20E strip and Biolog Gen III MicroPlate system. The 16S rRNA gene sequence of this strain was also obtained and used in strain identification and phylogenetic analysis. Healthy P. ridibundus larvae were challenged with RDL-2 and monitored to determine clinical signs consistent with the disease. Strain RDL-2 was identified as Aeromonas veronii based on its phenotypic and biochemical characteristics and on 16S rRNA gene sequence and phylogenetic analysis: RDL-2 was gram-negative, rod-shaped, and reacted positively for 69 (73%) of the 94 traits analysed; its 16S rRNA gene sequence displayed the highest homology to that of A. veronii bv. veronii (99.9%). Koch's postulates were fulfilled confirming that A. veronii is the causal agent of ulcerative syndrome. Thus, this study is the first to report A. veronii as a marsh frog pathogen in the Al-Ahsaa region. The aetiology of A. veronii as a potential poikilothermic pathogen shown here will expedite the development of diagnostic tests and methods for eradicating ulcerative syndrome.

Chromosome-Mediated mcr-3 Variants in Aeromonas veronii from Chicken Meat.

Two adjacent colistin resistance gene variants, termed mcr-3.3 and mcr-3-like, were identified in the chromosome of an Aeromonas veronii isolate obtained from retail chicken meat. The variants showed 95.20% and 84.19% nucleotide sequence identity, respectively, to mcr-3 from porcine Escherichia coli Functional cloning indicated that only mcr-3.3 conferred polymyxin resistance in both E. coli and Aeromonas salmonicida The mcr-3.3-mcr-3-like segment was also observed in other Aeromonas species, including A. media, A. caviae, and A. hydrophila.

Multidrug-Resistant Aeromonas veronii Recovered from Channel Catfish (Ictalurus punctatus) in China: Prevalence and Mechanisms of Fluoroquinolone Resistance.

To emphasize the importance of the appropriate use of antibiotics in aquaculture systems, the prevalence of resistance to 25 antimicrobials was investigated in 42 Aeromonas veronii strains isolated from farm-raised channel catfish in China in 2006-2012. All experiments were based on minimal inhibitory concentrations (MICs), and susceptibility was assessed according to the Clinical and Laboratory Standards Institute. Some isolates displayed antibiotic resistance to the latest-generation fluoroquinolones (i.e., ciprofloxacin, levofloxacin, and norfloxacin) in vitro. Therefore, we screened for genes conferring resistance to fluoroquinolones and performed conjugation experiments to establish the resistance mechanisms. The antibiotic resistance rates were 14.29-21.42% to three kinds of fluoroquinolones: ciprofloxacin, levofloxacin, and norfloxacin. Among the 42 strains isolated, 15 carried the qnrS2 gene. The MICs of the fluoroquinolones in transconjugants with qnrS2 were more than fourfold higher compared with the recipient. Among the fluoroquinolone-resistant A. veronii strains, eight had point mutations in both gyrA codon 83 (Ser83→Ile83) and parC codon 87 (Ser87→Ile87). However, five isolates with point mutations in parC codon 52 remained susceptible to the three fluoroquinolones. In conclusion, the mechanisms of fluoroquinolone resistance in A. veronii isolates may be related to mutations in gyrA codon 83 and parC codon 87 and the presence of the qnrS2 gene.