Effect of spatio-temporal variables, host fish species and on-farm biosecurity measures on the prevalence of potentially pathogenic Aeromonas species in freshwater fish farms.

AIMS: To determine the prevalence of Aeromonas species in freshwater fish farms, factors affecting their prevalence and virulence factors associated with each species. METHODS AND RESULTS: In a cross-sectional study from 128 farms in four districts of Uttar Pradesh, India, 11 species of Aeromonas were identified by gyrB sequencing including the first report of Aeromonas crassostreae from fish. Four species of Aeromonas were more prevalent (MP) in fish farms, A. veronii bv. sobria (50.0%) was the highest, followed by A. caviae (18.8%), A. veronii bv. veronii (11.7%) and A. dhakensis (7.0%). The less prevalent (LP) species were A. hydrophila, A. media, A. jandaei, A. allosaccharophila, A. salmonicida, A. crassostreae and A. taiwanensis. Spatial variation in the prevalence of Aeromonas species was observed. Dominance of biovar sobria ranged from 33.3 to 68.6%, notably lesser in farms with on-farm biosecurity measures. The prevalence of biovar veronii was significantly associated with pangas fish, rainy season and farms with on-farm biosecurity measures. The prevalence of LP species was significantly higher in mrigal fish and winter season. Multiple virulence factors (>6) were detected in 70.2% of the Aeromonas species. Significant association of ß-hemolysin, DNase, slime production, act, ahh1, aexT and lip was observed with different species of Aeromonas. Moreover, 75.8% of Aeromonas species possessed one or more enterotoxins genes (act/alt/ast). CONCLUSION: Significant association of spatio-temporal variables, host fish species and on-farm biosecurity measures were observed on the prevalence of some of the Aeromonas species in freshwater fish farms. Most of the Aeromonas species harboured virulence factors indicating their potential for pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study that determined the prevalence and identified the factors that affect the prevalence of Aeromonas species in freshwater fish farms. This information will be useful in managing Aeromonas infection in fish and their risks to public health.

Comparison of species, virulence genes and clones of Aeromonas isolates from clinical specimens and well water in Okinawa Prefecture, Japan.

AIMS: To reveal the sources of Aeromonas infection in Okinawa Prefecture of Japan, the species, virulence genes and clones of strains isolated from clinical specimens and well water were compared. METHODS AND RESULTS: The properties of both isolates were investigated by sequencing of rpoD, detection of 10 virulence genes using PCR and genotyping with pulsed-field gel electrophoresis. In all, 68 clinical and 146 well water strains of Aeromonas were isolated and the main species were A. caviae, A. dhakensis, A. hydrophila and A. veronii biovar sobria. Aeromonas dhakensis possessed various virulence genes; however, A. caviae possessed only fla. The same or similar clones were distributed in certain areas of Okinawa and one clone had survived several months in the biliary system of two patients, respectively. CONCLUSION: Although the same Aeromonas clone was not isolated from clinical and well water samples, our study revealed the detected patterns of virulence genes in both isolates, the distribution of identical/similar clones in the Okinawan environment and long-time survival in patient's organs. SIGNIFICANCE AND IMPACT OF THE STUDY: We investigated the association between Aeromonas patients and well water exposure. This study provides the properties of species, virulence genes and clones of Aeromonas isolated from samples of these origins.

Antimicrobial resistance in Aeromonas species isolated from aquatic environments in Brazil.

AIM: The current study was conducted to determine the antimicrobial resistance profile and genetic relatedness of Aeromonas sp. isolated from healthcare and urban effluents, wastewater treatment plant (WWTP) and river water. METHODS AND RESULTS: We detected the presence of genes conferring resistance to ß-lactam, quinolone and aminoglycoside. Multilocus sequence typing was carried out to differentiate the strains, and multilocus phylogenetic analysis was used to identify the species. A total of 28 cefotaxime-resistant Aeromonas sp. strains were identified, harbouring uncommon Guiana-extended-spectrum (GES)-type ß-lactamases (GES-1, GES-5, GES-7 and GES-16). Multidrug-resistant Aeromonas sp. were found in hospital wastewater, WWTP and sanitary effluent, and A. caviae was identified as the most prevalent species (85·7%). CONCLUSION: The release of untreated healthcare effluents, presence of antimicrobials in the environment, in addition to multidrug-resistant Aeromonas sp., are all potential factors for the spread of resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: We identified a vast repertoire of antimicrobial resistance genes (ARG) in Aeromonas sp. from diverse aquatic ecosystems, including those that encode enzymes degrading broad-spectrum antimicrobials widely used to treat healthcare-associated infections. Hospital and sanitary effluents serve as potential sources of bacteria harbouring ARG and are a threat to public health.

Structural-functional characterization of recombinant Apolipoprotein A-I fromLabeo rohitademonstrates heat-resistant antimicrobial activity.

Apolipoprotein A-I is an anti-inflammatory, antioxidative, cardioprotective, anti-tumorigenic, and anti-diabetic in mammals. Apolipoprotein A-I also regulates innate immune defense mechanisms in vertebrates and invertebrates. Apolipoproteins A-I from mammals and several teleosts display antibacterial activities against Gram negative and Gram positive bacteria. The present study describes strategies to obtain high amounts of soluble purified recombinant Apolipoprotein A-I of Labeo rohita, an Indian major carp (rLrApoA-I). The study also reports its detailed structural and functional characterization i.e. antimicrobial activity against a number of important marine and fresh water bacterial pathogens. The rLrApoA-I was expressed in Escherichia coli BL21(DE3) pLysS expression host as a soluble protein under optimized conditions. The yield of purified rLrApoA-I was ~ 75 mg/L from soluble fraction using metal ion affinity chromatography. The authenticity of the rLrApoA-I was confirmed by MALDI-TOF-MS analysis. The secondary structure analysis showed rLrApoA-I to be predominantly alpha helical, an evolutionary conserved characteristic across mammals and teleosts. The purified rLrApoA-I exhibited antimicrobial activity as evident from inhibition of growth of a number of bacteria namely Aeromonas hydrophila, A. liquefaciens, A. culicicola, A. sobria, Vibrio harveyi, V. parahaemolyticus and Edwardsiella tarda in a dose-dependent manner. Minimum bactericidal concentration for A. liquefaciens, A. culicicola, and A. sobria, was determined to be 25 µg/ml or 0.81 µM whereas for A. hydrophila, E. tarda, V. parahaemolyticus and V. harveyi, it was determined to be 100 µg/ml or 3.23 µM. These data strongly suggest that recombinant ApoA-I from Labeo rohita could play a role in primary defense against fish pathogen. Further, at temperature ≥ 55 °C, though a loss in secondary structure was observed, no effect on its antibacterial activity was observed. This is of significance as the antibacterial activity is not likely to be lost even if the protein is subjected to high temperatures during transport.

Population dynamics, antibiotics resistance and biofilm formation of Aeromonas and Vibrio species isolated from aquatic sources in Northern Malaysia.

This study aimed to compare population dynamics, antibiotic resistance and biofilm formation of Aeromonas and Vibrio species from seawater and sediment collected from Northern Malaysia. Isolates with different colony morphology were characterized using both biochemical and molecular methods before testing for antibiotic resistance and biofilm formation. Results obtained from this study showed that in Kedah, the population of Aeromonas isolated from sediment was highest in Pantai Merdeka (8.22 log CFU/ml), Pulau Bunting recorded the highest population of Aeromonas from sediment (8.43 log CFU/g). It was observed that Vibrio species isolated from seawater and sediment were highest in Kuala Sanglang (9.21 log CFU/ml). In Kuala Perlis, the population of Aeromonas isolated from seawater was highest in Jeti (7.94 log CFU/ml). Highest population of Aeromonas from sediment was recorded in Kampong Tanah Baru (7.99 log CFU/g). It was observed that Vibrio species isolated from seawater was highest in Padang Benta (8.42 log CFU/g) while Jeti Kuala Perlis had highest population of Vibrio isolated from sediment. It was observed that location does not influence population of Aeromonas. The results of the independent t - test revealed that there was no significant relationship between location and population of Vibrio (df = 10, t = 1.144, p > 0.05). The occurrence of biofilm formation and prevalence of antibiotic resistant Aeromonas and Vibrio species in seawater and sediment pose danger to human and aquatic animals' health.

Distribution of Aeromonas species in environmental water used in daily life in Okinawa Prefecture, Japan.

OBJECTIVES: The genus Aeromonas is known to causes diseases such as food poisoning, sepsis, and wound infection. However, the mode of Aeromonas transmission from environment to humans is not clearly understood. To evaluate the health risks of Aeromonas spp. in environmental freshwater, the number, proportion and putative virulence factors of Aeromonas species were investigated in Okinawa Prefecture, Japan. METHODS: Environmental freshwater samples were collected from three dams, two springs and three private wells. Aeromonas strains were identified by the biochemical method and the viable count was calculated. The production of extracellular enzymes and the virulence genes were investigated for possessing putative virulence factors using representative isolates. RESULTS: At least seven species of already-known Aeromonas isolates as well as unidentified Aeromonas spp. with/without arginin dehydrolase (ADH) exist in water at these sites. Aeromonas spp. was found to exist at over 1000 CFU/100 ml in one spring and two wells. A. veronii biovar sobria and A. jandaei were the predominant species in dams, and A. hydrophila and/or A. eucrenophila were predominant in wells. Almost all the sampled Aeromonas species produced protease, gelatinase, lipase, esterase and DNase, but A. caviae, A. caviae-like bacteria, and A. eucrenophila had low hemolytic activity. Most sampled A. hydrophila strains possessed both aerolysin gene (aer) and hemolysin gene (hlyA), but A. caviae and A. eucrenophila strains did not possess either gene. CONCLUSIONS: Since these results indicated that several Aeromonas species having potential pathogenicity exist in environmental water in Okinawa, surveys are recommended as a public health measure.

Changing epidemiological trend of Aeromonas species and ciprofloxacin sensitivity in South India.

Aeromonas species can cause a wide range of clinical infections. Several reports of drug resistance among the Aeromonas species have been reported, but our observations have differed. Here we present the changing susceptibility pattern of antibiotics for Aeromonas species over 14 years (January 2010-February 2024) at a tertiary care hospital in South India.

Bacteremia caused by Aeromonas species in patients with cancer: Clinical manifestations and outcomes.

INTRODUCTION: Oncologic patients can have severe infections due to Aeromonas. This study aims to investigate the clinical characteristics and outcomes of cancer patients with bloodstream infections (BSI) caused by Aeromonas. METHODOLOGY: We included patients with bacteremia caused by Aeromonas species from 2011 to 2018. RESULTS: Seventy-five BSI events in the same number of patients were identified. Forty patients were men (53.3%); the mean age was 49 years (IQR 28-61). A. caviae was the most frequent isolate (n = 29, 38.6%), followed by A. hydrophila (n = 23, 30.6%), A. sobria (n = 15, 20%), and A. veronii (n = 8, 10.6%). The most frequent underlying diagnosis was hematologic malignancy (n = 33, 44%), followed by breast cancer (n = 12, 16%) and gastrointestinal tract cancer (n = 8, 10.6%). The most frequent type of bacteremia was CRBSI in 32 cases (42.6%), followed by mucosal barrier injury-laboratory confirmed BSI (n = 20, 26.7%). Sixteen (26.2%) were hospital-acquired BSI. Attributable mortality occurred in 11 patients (14.6%). In univariate analysis A. hydrophila bacteremia, liver failure, skin/soft tissue infection, septic shock, inappropriate antimicrobial treatment, and relapse or cancer progression were associated with 30-day mortality. In multivariate analysis, only septic shock, inappropriate antimicrobial treatment, and relapse or cancer progression were associated with 30-day mortality. CONCLUSIONS: Aeromonas species should be considered one of the causative pathogens of healthcare-associated bacteremia, especially in immunocompromised patients. In addition, it can be associated with high fatality, particularly in patients with severe clinical infections.

Occurrence of Aeromonas Species in the Cutaneous Mucus of Barbour's Seahorses (Hippocampus barbouri) as Revealed by High-Throughput Sequencing.

Although several studies have described the bacterial community composition associated with marine fish, there is limited information related to seahorses. Moreover, previous studies have demonstrated that the skin microbiota is useful for determining health status and common disorders in the host. This study, therefore, aimed to explore the skin bacterial community composition in Barbour's seahorse (Hippocampus barbouri) using high-throughput sequencing of 16S ribosomal RNA genes. Water and sediment samples from the surrounding environment were also analyzed for comparative purposes. The results revealed that sequences affiliated with the Shewanellaceae family were dominant in the skin of female Barbour's seahorses and sediment samples, whereas sequences affiliated with the Bacillaceae family were dominant in the skin of male Barbour's seahorses. Interestingly, sequences affiliated with the Aeromonas genus were found in the skin of Barbour's seahorses, whose abundance was slightly similar between the female and male specimens. Further comparative analysis showed that the presence of Aeromonas species in the skin of Barbour's seahorses was strongly influenced by the surrounding sediment. Given that some Aeromonas species are known to be important pathogens in humans and fish, these results may be used for further research on the dependency of the skin microbial composition on the environment as well as determine whether the presence of Aeromonas and other detected species has implications on seahorse health.

Accurate Identification and Virulence Detection of Aeromonas: a Single-Center Retrospective Study on the Clinical Features and Outcomes Associated with Aeromonas Bacteremia in Southwestern China.

In this study, Aeromonas spp. were re-identified, and the clinical aspects associated with Aeromonas bacteremia, as well as drug resistance and virulence genes, were elucidated. A total of 188 isolates were classified into 7 Aeromonas spp. using housekeeping gene sequencing, which was the standard to assess the accuracy of the VITEK MALDI-TOF system and the VITEK2 Compact system. The VITEK MS system and housekeeping gene sequencing had a 39.89% clear coincidence rate, whereas the VITEK2 Compact system and the standard had a 2.13% coincidence rate. Aeromonas bacteremia was associated with septic shock, hematologic malignancy, and post-hepatitic cirrhosis. Hematological malignancy, hypoproteinemia, systemic steroid use, central venous catheterization, and virulence genes act and ast were linked to poor outcomes. Aeromonas bacteremia had a 37.5% mortality rate; however, differences in mortality rates among Aeromonas spp. were observed. According to the broth microdilution method, over 90% of isolates were sensitive to most antimicrobials, except ceftriaxone (83.33%) and imipenem (83.33%). Polymerase chain reaction and DNA sequencing verified the presence of drug resistance genes; blaCphA was detected in 3 isolates, while blaNDM-1 was found in one isolate. In summary, common methods for identifying Aeromonas spp. are ineffective. Immunocompromised patients have a higher risk of infection and mortality. Furthermore, carbapenem resistance is a serious problem.

Differences in flaA gene sequences, swimming motility, and biofilm forming ability between clinical and environmental isolates of Aeromonas species.

The flagellin A gene (flaA) sequences, swimming motility, and biofilm forming ability were investigated in order to reveal the genetic and functional differences of flagella between clinical and environmental isolates of Aeromonas species. Twenty-eight clinical and 48 environmental strains of Aeromonas species isolated in Okinawa Prefecture of Japan were used in this study. The full-length flaA genes of these strains were sequenced and aligned, and a phylogenetic tree was constructed. In addition, swimming motility and biofilm forming ability were evaluated by conventional methods. Aeromonas veronii biovar sobria and A. hydrophila clearly divided into clinical and environmental strain clusters in the flaA phylogenetic classification, and the six and 13 specific amino acids respectively, of FlaA of both species were different in clinical and environmental strains. Furthermore, the flaA size of the clinical strain of A. veronii bv. sobria was mainly 909, 924, and 939 bp, and the size of A. hydrophila was 909 bp. The swimming motility of clinical isolates of both species was lower than the environmental isolates; however, the biofilm forming ability of the clinical isolates was high. Thus, the clinical isolates of A. veronii bv. sobria and A. hydrophila had different genetic and functional characteristics of flagellin than the environmental isolates. The characteristics of flagellin could serve as indicators to distinguish between clinical and environmental isolates of the both species. It may contribute to diagnosis of these diseases and the monitoring of clinical strain invasion into the natural environment.

Bacteriophages in the Control of Aeromonas sp. in Aquaculture Systems: An Integrative View.

Aeromonas species often cause disease in farmed fish and are responsible for causing significant economic losses worldwide. Although vaccination is the ideal method to prevent infectious diseases, there are still very few vaccines commercially available in the aquaculture field. Currently, aquaculture production relies heavily on antibiotics, contributing to the global issue of the emergence of antimicrobial-resistant bacteria and resistance genes. Therefore, it is essential to develop effective alternatives to antibiotics to reduce their use in aquaculture systems. Bacteriophage (or phage) therapy is a promising approach to control pathogenic bacteria in farmed fish that requires a heavy understanding of certain factors such as the selection of phages, the multiplicity of infection that produces the best bacterial inactivation, bacterial resistance, safety, the host's immune response, administration route, phage stability and influence. This review focuses on the need to advance phage therapy research in aquaculture, its efficiency as an antimicrobial strategy and the critical aspects to successfully apply this therapy to control Aeromonas infection in fish.

Prevalence of antimicrobial resistance in bacteria isolated from Great Cormorants (Phalacrocorax carbo hanedae) in Japan.

Wild birds are recognized as disseminators of antimicrobial-resistant (AMR) bacteria into the environment. Here, we isolated AMR indicator bacteria from 198 Great Cormorant cloacal swabs collected in Shiga (n=90), Oita (n=52), Gifu (n=29), and Gunma (n=27) Prefectures, Japan, in 2018 and 2019. In total, 198 Aeromonas spp. and 194 Escherichia spp. were isolated, and their antimicrobial susceptibility was examined. Aeromonas spp. were resistant to colistin (8.6%), nalidixic acid (4%), and other antimicrobials (<2%), with 3.0% positivity for mcr-3. Escherichia spp. showed resistance to colistin (3.1%), ampicillin (2.6%), tetracycline (2.1%), and other antimicrobials (<2%). This study shows the presence of AMR bacteria in Great Cormorants, indicating that these birds potentially disseminate AMR bacteria.

Molecular and Phenotypic Analysis of Hemolytic Aeromonas Strains Isolated from Food in Egypt Revealed Clinically Important Multidrug Resistance and Virulence Profiles.

The aim of this study was to determine the public health significance of hemolytic Aeromonas species isolated from 213 food samples in Egypt, based on their virulence and antimicrobial-resistance potential. We recovered 63 strains, isolated from fish, raw milk, karish cheeses, and ras cheese in 29 (31.18%) of 93, 10 (25.00%) of 40, 13 (32.50%) of 40, and 11 (27.50%) of 40 samples, respectively. The most prevalent virulence gene was alt (50.79%), followed by aerA (34.92%), asa1 (39.68%), ahh1 (20.63%), act (11.11%), and ast (3.17%). Thirteen strains screened in this study carried no hemolysin gene, but only the alt gene, and another eight hemolytic strains screened, carried no virulence gene. The virulence signatures " ahh1+ aerA" and " alt+ act," in which the genes interact synergistically to induce severe diarrhea, were detected in two and four strains, respectively. Most showed resistance to third-generation cephalosporins, aztreonam, and imipenem, which indicates the complexity of the ß-lactamase production in our hemolytic Aeromonas strains. Fourteen (22.22%) of 63 strains carried one or more antimicrobial-resistance markers, including the blaCTX-M, blaTEM, tet(A), tet(E), and intI1 genes, which were detected in 6.34, 3.17, 3.17, 4.76, and 14.28% of isolates, respectively. In conclusion, the majority of hemolytic Aeromonas strains isolated from the intestinal contents of healthy fish and naturally contaminated milk and cheeses were not commensal but had developed multidrug-resistance and virulence profiles, indicating an emerging potential health risk. Importantly, screening for certain hemolysin genes may not be reliable in predicting the pathogenic potential of Aeromonas species and, thereby, the safety of analyzed foods. Our findings indicate that specific criteria are required for the phenotypic and molecular analysis of Aeromonas species in food items, particularly those eaten without further treatment, to ensure their safety.

Potential enterotoxicity and antimicrobial resistance pattern of Aeromonas species isolated from pet turtles and their environment.

To investigate the potential enterotoxicity and antimicrobial resistance of aeromonads from pet turtles as a risk for human infection, one hundred and two Aeromonas spp. were isolated from the feces, skin and rearing environments of pet turtles and identified by biochemical and gyrB sequence analyses. Aeromonas enteropelogenes was the predominant species among the isolates (52.9%) followed by A. hydrophila (32.4%), A. dharkensis (5.9%), A. veronii (4.9%) and A. caviae (3.9%). Their potential enterotoxicities were evaluated by PCR assays for detecting genes encoding cytotoxic enterotoxin (act) and two cytotonic enterotoxins (alt and ast). 75.8% of A. hydrophila isolates exhibited the act+/alt+/ast+ genotype, whereas 94.4% of A. enteropelogenes isolates were determined to be act-/alt-/ast-. In an antimicrobial susceptibility test, most isolates were susceptible to all tested antibiotics except amoxicillin, ampicillin, cephalothin, chloramphenicol and tetracycline. Non-susceptible isolates to penicillins (ampicillin and amoxicillin) and fluoroquinolones (ciprofloxacin and norfloxacin) were frequently observed among the A. enteropelogenes isolates. Few isolates were resistant to imipenem, amikacin, ceftriaxone and cefotaxime. Collectively, these results suggest that pet turtles may pose a public health risk of infection by enterotoxigenic and antimicrobial resistant Aeromonas strains.

Sanitary evaluation of domestic water supply facilities with storage tanks and detection of Aeromonas, enteric and related bacteria in domestic water facilities in Okinawa Prefecture of Japan.

To provide for temporary restrictions of the public water supply system, storage tanks are commonly installed in the domestic water systems of houses and apartment buildings in Okinawa Prefecture of Japan. To learn more about the sanitary condition and management of these water supply facilities with storage tanks (hereafter called "storage tank water systems") and the extent of bacterial contamination of water from these facilities, we investigated their usage and the existence of Aeromonas, enteric and related bacteria. Verbal interviews concerning the use and management of the storage tank water systems were carried out in each randomly sampled household. A total of 54 water samples were collected for bacteriological and physicochemical examinations. Conventional methods were used for total viable count, fecal coliforms, identification of bacteria such as Aeromonas, Enterobacteriaceae and non-fermentative Gram-negative rods (NF-GNR), and measurement of residual chlorine. On Aeromonas species, tests for putative virulence factor and an identification using 16S rRNA and rpoB genes were also performed. Water from the water storage systems was reported to be consumed directly without boiling in 22 of the 54 houses (40.7%). 31 of the sampled houses had installed water storage tanks of more than 1 cubic meter (m3) per inhabitant, and in 21 of the sampled houses, the tank had never been cleaned. In all samples, the total viable count and fecal coliforms did not exceed quality levels prescribed by Japanese waterworks law. Although the quantity of bacteria detected was not high, 23 NF-GNR, 14 Enterobacteriaceae and 5 Aeromonas were isolated in 42.6%, 7.4% and 3.7% of samples respectively. One isolated A. hydrophila and four A. caviae possessed various putative virulence factors, especially A. hydrophila which had diverse putative pathogenic genes such as aer, hlyA, act, alt, ast, ser, and dam. Many bacteria were isolated when the concentration of residual chlorine was below 0.1 mg/l and the water temperature was above 20 °C. These results suggest that elevated water temperature and mismatch between tank size and water demand lead to loss of residual chlorine in tap water. Therefore, to minimize growth of aquatic bacteria such as Aeromonas spp. and Pseudomonas spp., we recommend that an appropriate size tank and/or volume of stored water is always used, and also suggest installation of some means of reducing water temperature such as shading.

Design of an Immersion Vaccine Against Aeromonad Septicemia in Perch (Perca fluviatilis L.).

The production of an immersion vaccine and the vaccination procedure to immunize fry of perch (Perca fluviatilis L.) against pathogenic Aeromonas sobria that harbor a type III secretion system is described. The vaccine, based on chemically inactivated A. sobria, enables rapid vaccination of a large number of fish by immersion of fry in an aqueous vaccine suspension during 5 min, giving them high protection during fattening under open water conditions in a freshwater lake for at least 4 months.