Hydrophila Isolates Research Articles

Assessment of carbapenem resistance and carbapenemase genes in wastewater from cattle slaughterhouses: Implications for environmental antibiotic resistance surveillance

The objectives of the study were to determine the prevalence of carbapenem-resistant Gram-negative bacteria and assess the potential risks associated with cattle slaughterhouse wastewater. A total of 270 wastewater samples were collected from 10 different cattle slaughterhouses for microbiological analysis. Conventional culture methods were employed, followed by disc diffusion, the Modified Carbapenem Inactivation Method (mCIM), and the Modified Hodge Test (MHT) to identify carbapenem resistance. The Vitek® 2 compact system was used for species identification and antibiotic susceptibility profiling. Conventional and quantitative PCR (qPCR) were performed to detect specific carbapenemase genes (blaKPC, blaNDM, and blaOXA-48). Among the collected 72 carbapenem-resistant isolates, one Pseudomonas fluorescens, one Aeromonas hydrophila, and two Aeromonas sobria exhibited resistance to meropenem. Additionally, six P. fluorescens and two A. hydrophila isolates demonstrated intermediate resistance to meropenem. Furthermore, five carbapenem-resistant isolates were identified as Stenotrophomonas maltophilia, known to be inherently resistant to most antibiotics. Ten different antibiotics were evaluated in the antibiotic resistance panel and all Aeromonas isolates were found to be resistant to cefazolin and one A. hydrophila was detected as multi-drug resistant. The revealed data indicates that slaughterhouse wastewater can serve as a reservoir for antibiotic-resistant opportunistic pathogens. However, it may not pose a substantial risk for the distribution of carbapenemases, thereby mitigating concerns related to potential public health and environmental hazards associated with this aspect of slaughterhouse operations. This study contributes to understanding of antibiotic resistance in livestock-related environments and underscores the importance of continued monitoring and surveillance.

Watery diarrhea between Vibrio cholera and Aeromonas hydrophila during Iraqi cholera outbreaks

Cholera remains a significant health challenge globally, especially in regions with poor infrastructure and healthcare. This study aimed to detect Aeromonas hydrophila in clinical samples from diarrhea patients during the 2022-2023 outbreaks in Iraq. Archived samples from Baquba General Teaching Hospital were analyzed using culture media, biochemical tests, and the VITEK 2 system. Both Vibrio cholerae and A. hydrophila isolates produced virulence factors such as hemolysin, protease, lipase, phospholipase, and biofilm. Antibiotic susceptibility testing showed A. hydrophila was susceptible to cefepime, ceftazidime, amikacin, imipenem, meropenem, and piperacillin-tazobactam but resistant to aztreonam, levofloxacin, and ciprofloxacin. V. cholerae was susceptible to amikacin, imipenem, meropenem, piperacillin-tazobactam, doxycycline, and chloramphenicol but resistant to trimethoprim-sulfamethoxazole and ampicillin. The findings highlight the need for better recognition of Aeromonas as a gastrointestinal pathogen and emphasize the importance of improved diagnostics and public health initiatives to reduce diarrheal diseases. Highlight: Detection Methods: Culture media, biochemical tests, VITEK 2 system used. Virulence Factors: Hemolysin, protease, lipase, phospholipase, biofilm production identified. Antibiotic Resistance: Specific resistance observed in A. hydrophila and V. cholerae. Keyword: Cholera, Aeromonas hydrophila, Antibiotic Susceptibility, Diarrhea, Iraq Outbreak

Characterisation, prevalence and antibiogram of Aeromonas hydrophila in turtles: insights into virulence factors, antimicrobial resistance genes and innate immunity

ABSTRACT Bacterial infection poses a significant threat to the health and survival of turtles, both in their natural habitats and in captivity. Notably, the presence and interactions of bacteria, such as those belonging to the Aeromonas genus, have been identified for their adverse impact on turtle health and ecosystem dynamics. This study investigated the prevalence, antibiotic susceptibility, virulence factors, and antimicrobial resistance genes of Aeromonas hydrophila in Trachemys scripta elegans (Red-eared Slider) and Chelonia mydas (Green Turtle). The study also examined the innate immunity of T. scripta elegans and C. mydas. Buccal and cloacal swabs from 39 turtles were collected and examined alongside 45 water samples to try to isolate and amplify A. hydrophila. Polymerase chain reaction (PCR) amplifications were then sequenced to identify the presence of specific genes associated with virulence, and antimicrobial resistance. The findings revealed that A. hydrophila had a prevalence rate of 35.9% in all examined turtle species, with 14 water samples accounting for 31% of the total samples. Buccal samples from Green and Red-eared turtles showed a higher rate of A. hydrophila isolates than cloacal samples. Sensitivity to norfloxacin (85.71%), gentamycin (71.42%), and ceftriaxone (64.28%) was observed, whereas resistance to amoxicillin (64.28%), ampicillin (57.14%), and rifamycin (57.14%) was apparent. Antibiotic resistance genes, including blaTEM and blaSHV, in A. hydrophila isolates, were present in 42.86% and 28.5% of the samples, respectively. PCR analysis confirmed the genetic identity of A. hydrophila, and five virulence genes were detected: AHCYTOEN (100%), followed by aerolysin (92.8%), alt and act (85.7%), and lipase (78.5%). Genetic sequences of the isolates had a 99% identity with the reference strains in GenBank. Furthermore, the study found the innate immunity of turtles to be robust due to serum lysozyme activity, bactericidal killing assay (BKA), total leukocyte count, heterophil/lymphocyte ratio, and packed cell volume (PCV%), all of which help to protect them from infections. Overall, this research highlights the need for continued monitoring and management of antibiotic resistance in aquatic ecosystems.

Distribution of bacteria and antimicrobial resistance in retail Nile tilapia (Oreochromis spp.) as potential sources of foodborne illness.

This study aimed to investigate AMR profiles of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from Nile tilapia (Oreochromis spp.) (n = 276) purchased from fresh markets and supermarkets in Bangkok, Thailand. A sample of tilapia was divided into three parts: fish intestine (n = 276), fish meat (n = 276), and liver and kidney (n = 276). The occurrence of A. hydrophila, Salmonella, and V. cholerae was 3.1%, 7.4%, and 8.5%, respectively. A high prevalence of these pathogenic bacteria was observed in fresh market tilapia compared to those from supermarkets (p < 0.05). The predominant Salmonella serovars were Paratyphi B (6.4%), followed by Escanaba (5.7%), and Saintpaul (5.7%). All isolates tested positive for the virulence genes of A. hydrophila (aero and hly), Salmonella (invA), and V. cholerae (hlyA). A. hydrophila (65.4%), Salmonella (31.2%), and V. cholerae (2.9%) showed multidrug resistant isolates. All A. hydrophila isolates (n = 26) exhibited resistant to ampicillin (100.0%) and florfenicol (100.0%), and often carried sul1 (53.8%) and tetA (50.0%). Salmonella isolates were primarily resistant to ampicillin (36.9%), with a high incidence of blaTEM (26.2%) and qnrS (25.5%). For V. cholerae isolates, resistance was observed against ampicillin (48.6%), and they commonly carried qnrS (24.3%) and tetA (22.9%). To identify mutations in the quinolone resistance determining regions (QRDRs), a single C248A point mutation of C248A (Ser-83-Tyr) in the gyrA region was identified in six out of seven isolates of Salmonella isolates. This study highlighted the presence of antimicrobial-resistant pathogenic bacteria in Nile tilapia at a selling point. It is important to rigorously implement strategies for AMR control and prevention.

Molecular characterization of Aeromonas hydrophila detected in Channa marulius and Sperata sarwari sampled from rivers of Punjab in Pakistan.

Aeromonas hydrophila is one of the major pathogenic bacteria responsible for causing severe outbreaks at fish farms and is also a major global public health concern. This bacterium harbors many virulence genes. The current study was designed to evaluate the antidrug and virulence potential of A. hydrophila by amplifying its antimicrobial resistance and virulence genes using PCR and examining their effects on fish tissues and organs. A total of 960 fish samples of Channa marulius and Sperata sarwari were collected from four sites of the rivers of the Punjab, Pakistan. A. hydrophila isolates were subjected to biochemical identification and detection of virulence and antimicrobial resistance (AMR) genes by PCR. We retrieved 181 (6.46%) A. hydrophila isolates from C. marulius and 177 (6.25%) isolates from S. sarwari. Amplification through PCR revealed the incidence of virulence genes in 95.7% of isolates in C. marulius and 94.4% in S. sarwari. Similarly, amplification through PCR also revealed occurrence of AMR genes in 87.1% of isolates in C. marulius and 83.9% in S. sarwari. Histopathological examination revealed congestion (5.2%) and hepatocyte necrosis (4.6%) in liver, lamellar fusion (3.3%) and the presence of bacterial colonies (3.7%) in gills, fin erosion (6%), and the presence of biofilms (3.5%) in tail fins of infected fish. Phylogenetic tree analysis of 16S rRNA and gyrB gene of A. hydrophila revealed 100% and 97% similarity, respectively, with 16S rRNA gene and gyrB of A. hydrophila isolated in previous studies. The results of antimicrobial susceptibility testing showed that all isolates demonstrated resistance to sulfamethoxazole, ampicillin, neomycin, and norfloxacin, while susceptibility to gentamicin, chloramphenicol, and tetracycline, and intermediate resistance was observed against cefotaxime. The results concluded that examined fish samples were markedly contaminated with virulent and multidrug strains of A. hydrophila which may be of a potential health risk. The study emphasizes the responsible antimicrobial use in aquaculture and the urgent need for effective strategies to control the spread of virulence and antimicrobial resistance genes in A. hydrophila.

Phenotypic, molecular detection, and Antibiotic Resistance Profile (MDR and XDR) of Aeromonas hydrophila isolated from Farmed Tilapia zillii and Mugil cephalus

In the present study, Aeromonas hydrophila was isolated from Tilapia zillii and Mugil cephalus samples collected during different seasons from various Suez Canal areas in Egypt. The prevalence of A. hydrophila, virulence genes, and antibiotic resistance profile of the isolates to the commonly used antibiotics in aquaculture were investigated to identify multiple drug resistance (MDR) and extensive drug-resistant (XDR) strains. In addition, a pathogenicity test was conducted using A. hydrophila, which was isolated and selected based on the prevalence of virulence and resistance genes, and morbidity of natural infected fish. The results revealed that A. hydrophila was isolated from 38 of the 120 collected fish samples (31.6%) and confirmed phenotypically and biochemically. Several virulence genes were detected in retrieved A. hydrophila isolates, including aerolysin aerA (57.9%), ser (28.9%), alt (26.3%), ast (13.1%), act (7.9%), hlyA (7.9%), and nuc (18.4%). Detection of antibiotic-resistant genes revealed that all isolates were positive for blapse1 (100%), blaSHV (42.1%), tetA (60.5%), and sul1 (42.1%). 63.1% of recovered isolates were considered MDR, while 28.9% of recovered isolates were considered XDR. Some isolates harbor both virulence and MDR genes; the highest percentage carried 11, followed by isolates harboring 9 virulence and resistance genes. It could be concluded that the high prevalence of A. hydrophila in aquaculture species and their diverse antibiotic resistance and virulence genes suggest the high risk of Aeromonas infection and could have important implications for aquaculture and public health.

First Report and Characterization of a Plasmid-Encoded blaSFO-1 in a Multi-Drug-Resistant Aeromonas hydrophila Clinical Isolate.

Antibiotic resistance remains one of the most pressing public health issues facing the world today. At the forefront of this battle lies the ever-increasing identification of extended-spectrum beta-lactamases and carbapenemases within human pathogens, conferring resistance towards broad-spectrum and last-resort antimicrobials. This study was prompted due to the identification of a pathogenic Aeromonas hydrophila isolate (strain MAH-4) collected from abdominal fluid, which presented a robust resistance pattern against second-, third-, and fourth-generation cephalosporins, ertapenem, ciprofloxacin, gentamicin, levofloxacin and moxifloxacin, and beta lactam/beta-lactamase inhibitor combinations. Whole genome sequencing was performed and identified a 328 kb plasmid (pMAH4) encoding 10 antibiotic resistance genes, including blaSFO-1, blaTEM-1, and blaOXA-1 of A. hydrophia MAH-4. This is the first report of beta-lactamase SFO-1 within a clinical strain of Aeromonas. Due to the remarkable sequence identity of pMAH4 to plasmids associated with Enterobacterales genera like Klebsiella and the extensive capabilities of Aeromonas for horizontal gene transfer, our identification of a clinical isolate encoding SFO-1 on a plasmid suggests antibiotic resistance gene mobility between Enterobacterales and non-Enterobacterales species.

Deteksi Gen Resistensi Aeromonas hydrophila yang Diisolasi dari Budidaya Ikan Lele Di Jambi

Aeromonas hydrophila is one of the causative agents for Motile Aeromonas Septicemia (MAS) in catfish. MAS treatment is generally carried out by administering antibiotics. This study aimed to determine the antibiotic resistance profile of A. hydrophila and detect the presence of resistance genes. The samples are archived A. hydrophila isolates isolated from catfish ponds in the district of Muara Jambi, Bungo, Jambi Province, and Merangin total of 18 isolates. Testing the antibiotic resistance profile was carried out using the disk diffusion method against tetracycline, oxytetracycline, ampicillin, amoxicillin, ciprofloxacin, enrofloxacin, and streptomycin. The detected resistance genes were ampC, qnrA, qnrB, and qnrS. The results showed A. hydrophila isolates were resistant to tetracycline (100%), oxytetracycline (94.42%), ampicillin (77.70%), and amoxicillin (77.72%) and still sensitive to ciprofloxacin (83.32%), enrofloxacin (100 %), and streptomycin (100%). The ampC gene was found in 8 out of 14 isolates resistant to beta-lactam antibiotics (57.14%). The qnrA and qnrS genes were found in 3 out of 4 isolates resistant to quinolone antibiotics (75%), while qnrB was found in 1 isolate (25%). A. hydrophila isolated from catfish ponds in Jambi develops resistance to antibiotics and resistance genes found in isolates resistant to antibiotics.

Cross-sectional analysis of risk factors associated with Mugil cephalus in retail fish markets concerning methicillin-resistant Staphylococcus aureus and Aeromonas hydrophila.

Aeromonas hydrophila and methicillin-resistant Staphylococcus aureus (MRSA) are potent bacterial pathogens posing major hazards to human health via consuming fish harboring these pathogens or by cross-contamination beyond the contaminated environment. The aim of this study was to determine risk variables associated with the presence of certain pathogenic bacteria from Mugil cephalus fish in retail markets in Egypt. The virulence genes of A. hydrophila and S. aureus were also studied. Furthermore, the antibiotic sensitivity and multidrug resistance of the microorganisms were evaluated. In a cross-sectional investigation, 370 samples were collected from mullet skin and muscle samples, washing water, fish handlers, knives, and chopping boards. Furthermore, fish handlers' public health implications were assessed via their response to a descriptive questionnaire. S. aureus and Aeromonas species dominated the investigated samples with percentages of 26.76% and 30.81%, respectively. Furthermore, A. hydrophila and MRSA were the predominant recovered bacterial pathogens among washing water and knives (53.85% and 46.66%, respectively). The virulence markers aerA and hlyA were found in 90.7% and 46.5% of A. hydrophila isolates, respectively. Moreover, the virulence genes nuc and mec were prevalent in 80% and 60% of S. aureus isolates, respectively. Antimicrobial susceptibility results revealed that all A. hydrophila isolates were resistant to amoxicillin and all MRSA isolates were resistant to amoxicillin and ampicillin. Remarkably, multiple drug resistance (MDR) patterns were detected in high proportions in A. hydrophila (88.37%) and MRSA (100%) isolates. The prevalence of Aeromonas spp. and S. aureus had a positive significant correlation with the frequency of handwashing and use of sanitizer in cleaning of instruments. MRSA showed the highest significant prevalence rate in the oldest age category. The pathogenic bacteria recovered in this study were virulent and had a significant correlation with risk factors associated with improper fish handling. Furthermore, a high frequency of MDR was detected in these pathogenic bacteria, posing a significant risk to food safety and public health.

Molecular characterization of Aeromonas hydrophila isolates from diseased fishes in district Kasur, Punjab, Pakistan

Abstract Pakistan is an agricultural country and fisheries play a very important role in the economic development of the country. Different diseases are prevalent in Pakistani fish but information related to the causative agents is not well-known. Keeping in view the significance of bacterial pathogens as the causative agents of multiple fish diseases, the present study was conducted for identification, characterization and analysis of virulence genes of Aeromonas spp. isolated from diseased fishes. A total of fifty fish samples having multiple clinical indications were collected from different fish farms of district Kasur, Punjab Pakistan. For isolation of Aeromonas spp. samples were enriched and inoculated on Aeromonas isolation medium. Isolates were identified and characterized by different biochemical tests, Analytical Profile Index (API) 20E kit and Polymerase Chain Reaction (PCR) assays. All isolates were screened for three putative virulence genes including aerolysin (aer), haemolysin (hyl) and heat labile cytotonic enterotoxin (alt). Seven isolates of Aeromonas (A.) hydrophila were retrieved and identified based on API 20E. These isolates were further confirmed as A. hydrophila on the basis of PCR assays. Three isolates were detected positive for the presence of virulence genes (alt and hyl). Whereas aerolysin (aer) gene was not present in any of A. hydrophila isolates. The present study confirmed A. hydrophila as the causative agent of epizootic ulcerative syndrome and motile Aeromonas septicemia in fish farms of district Kasur, Punjab Pakistan. Moreover, detection of two virulence genes (alt and hyl) in A. hydrophila isolates is a threat for fish consumers of study area.

Investigation of Cephalosporin and Heavy Metal Resistance of Aeromonas hydrophila and Pseudomonas aeruginosa Strains Isolated from Hospital Sewage in Türkiye

In this study, 89 strains of Aeromonas hydrophila and 88 strains of Pseudomonas aeruginosa were isolated from the sewage of a university hospital in Turkey. The resistance of these bacterial isolates to 11 different cephalosporin classes’ antibiotics belonging to four generations and to 4 heavy metals was investigated. Cadmium, lead, manganese, and zinc are the heavy metals employed. There was a high incidence of resistance to cefazolin (98.9%), cefaclor (98.9%), and cefprozil (97.8%) among the A. hydrophila isolates. Lower resistance to cefoxitin (30.3%), cefepime (30.3%), and ceftazidime (31.4%) were found. Cefazolin, cefuroxime, cefaclor, and cefoxitin resistance was found to be (100%) among the P. aeruginosa isolates. Moreover, resistance rates to cefprozil (98.9%), cefixime (96.6%), and ceftizoxime (85.2%) were detected. No isolates of P. aeruginosa were showed resistance to ceftazidime, cefepime and cefpirome. Multiple antibiotic resistance (MAR) indexes ranged from 0.27 to 1.0 among A. hydrophila isolates and from 0.46 to 0.72 among P. aeruginosa isolates. All of the A. hydrophila and P. aeruginosa isolates showed resistance to cadmium. A. hydrophila and P. aeruginosa isolates showed low resistance to lead of 6.7% and 2.2% respectively. Results indicate that both species are easily recovered in hospital sewage and these species gained resistance to different generations of cephalosporins and heavy metals.

Pathogenic Aeromonas spp in Amazonian fish: Virulence genes and susceptibility in Piaractus brachypomus, the main native aquaculture species in Peru

The aim of this study was to isolate and identify Aeromonas species, characterize their virulence genes factors, and to evaluate their lethality in Piaractus brachypomus. Presumptive Aeromonas (n = 34) were selected from 44 bacteria retrieved from diseased Amazonian fish. Nineteen isolates were chosen based on fish species, group, the location where were isolated, and colony morphology. Isolates were biochemically and molecularly identified as Aeromonas hydrophila, A. dhakensis, A. veronii, A. jandaei, and A. caviae using API 20E, API 20NE and the analysis of 16 S rRNA and gyrB genes, respectively. Eight virulences genes (aer, altA, act, hly, ser, lip, fla ahyB) were found in one A. dhakensis and two A. hydrophila isolates. A. veronii, A. jandaei, and another two A. hydrophila isolates harbored four, two and three virulence genes, respectively. All A. caviae were fla +, ahyB + and lip +, except two isolates that also presented ser and altA. P. brachypomus (9 ± 1 cm), infected intraperitoneally (4 ×106, 4 ×107, and 4 ×108 CFU/fish), showed susceptibility to all Aeromonas species, each harboring a different virulence genes profile. Both A. hydrophila and A. dhakensis caused > 80% mortality at the lowest evaluated concentration (∼106 CFU/fish). A. veronii and A. jandaei isolates produced 20% and 70% mortality, respectively, using ∼107 CFU/fish, but with lower intensity compared to A. hydrophila and A. dhakensis. At the highest concentration (∼108 CFU/fish), A. veronii caused 70% mortality, while A. caviae isolates caused mortality ranging from 50% to 80%. Remarkably, A. jandei resulted in 100% mortality at this level, similar to A. hydrophila and A. dhakensis. This report confirms the variety of Aeromonas species infecting farmed native Amazonian fish. The study also suggests that virulence genes evaluated in Aeromonas species may not always be an indicator of virulence. Moreover, P. brachypomus, an economically important aquaculture fish species could be susceptible not only to the high mentioned A. hydrophila but also to other less observed species such as A. dhakensis, A. veronii, A. jandaei, and A. caviae, bacteria that should be taken into account in health surveillance for the implementation of aquaculture disease prevention strategies.

Comparative genomic analysis of Aeromonas dhakensis and Aeromonas hydrophila from diseased striped catfish fingerlings cultured in Vietnam.

Motile Aeromonas septicemia (MAS) is a burden for striped catfish (Pangasius hypophthalmus) farmers in Vietnam. MAS can be caused by several species of Aeromonas but Aeromonas hydrophila is seen as the leading cause of MAS in aquaculture, but recent reports suggest that A. dhakensis is also causing MAS. Here we investigated the bacterial etiology of MAS and compared the genomic features of A. hydrophila and A. dhakensis. We collected 86 isolates from diseased striped catfish fingerlings over 5 years from eight provinces in Vietnam. Species identification was done using PCR, MALDI-TOF and whole genome sequence (WGS). The MICs of commonly used antimicrobials was established. Thirty presumed A. hydrophila isolates were sequenced for species confirmation and genomic comparison. A phylogenetic analysis was conducted using publicly available sequences and sequences from this study. A total of 25/30 isolates were A. dhakensis sequence type (ST) 656 and 5/30 isolates were A. hydrophila ST 251. Our isolates and all publicly available A. hydrophila isolates from Vietnam belonged to ST 251 and differed with <200 single nucleotide polymorphisms (SNP). Similarly, all A. dhakensis isolates from Vietnam belonged to ST 656 and differed with <100 SNPs. The tet(A) gene was found in 1/5 A. hydrophila and 19/25 A. dhakensis. All A. hydrophila had an MIC ≤2 mg/L while 19/25 A. dhakensis had MIC ≥8 mg/L for oxytetracycline. The floR gene was only found in A. dhakensis (14/25) which showed a MIC ≥8 mg/L for florfenicol. Key virulence genes, i.e., aerA/act, ahh1 and hlyA were present in all genomes, while ast was only present in A. dhakensis. This study confirms previous findings where A. dhakensis was the dominating pathogen causing MAS and that the importance of A. hydrophila has likely been overestimated. The differences in antimicrobial susceptibility between the two species could indicate a need for targeted antimicrobial treatment plans. The lipopolysaccharide regions and outer membrane proteins did not significantly differ in their immunogenic potentials, but it remains to be determined with in vivo experiments whether there is a difference in the efficacy of available vaccines against A. hydrophila and A. dhakensis.

Phenotypic, molecular detection and antibiogram analysis of Aeromonas Hydrophila from Oreochromis Niloticus (Nile Tilapia) and Ready-To- eat fish products in selected Rift Valley lakes of Ethiopia

BackgroundAeromonas hydrophila is a zoonotic bacterial pathogen that frequently causes disease and mass mortalities among cultured and feral fishes worldwide. In Ethiopia, A. hydrophila outbreak was reported in Sebeta fish ponds and in Lake Tana fishery. However, there is no to little information on the molecular, and phenotypical characteristics of A. hydrophila in Ethiopian fisheries. Therefore, a cross-sectional study was conducted from November 2020 to May 2021 in selected Ethiopian Rift valley lakes.ResultsA total of 140 samples were collected aseptically from fish (Muscle, Gill, Intestine, Spleen and Kidney) from fish landing sites, market and restaurants with purposive sampling methods. Aeromonas selective media (AMB), morphological and biochemical tests were used to isolate and identify A. hydrophila. Accordingly, the pathogen was isolated from 81 (60.45%) of samples. Among the isolates 92.59% expressed virulence trait through β hemolysis on blood agar media with 5% sheep blood. Moreover, 54 strains (66.67%) were further confirmed with Real-Time PCR (qPCR) using ahaI gene specific primers and optimized protocol. The highest (68.51%) were detected from live fish, (24.07%) were from market fish and the lowest (7.4%%) were from ready-to-eat products. Antibiogram analysis was conducted on ten representative isolates. Accordingly, A. hydrophila isolates were susceptible to ciprofloxacin (100%), chloramphenicol (100%) and ceftriaxone (100%). However, all ten isolates were resistant to Amoxicillin and Penicillin.ConclusionsThe study indicates A. hydrophila strains carrying virulence ahaI gene that were ß-hemolytic and resistant to antibiotics commonly used in human and veterinary medicine are circulating in the fishery. The detection of the pathogen in 140 of the sampled fish population is alarming for potential outbreaks and zoonosis. Therefore, further molecular epidemiology of the disease should be studied to establish potential inter host transmission and antibiotic resistance traits. Therefore, raising the public awareness on risk associated with consuming undercooked or raw fish meat is pertinent.

Effect of aquaponic water and substratum material on biofilm formation by Aeromonas hydrophila

Aeromonas hydrophila is a zoonotic pathogen causing illness in fish and susceptible humans. This emerging pathogen has been isolated within aquaponic systems and could cause disease in fish and a hazard to humans consuming aquaponic produce. This study determined whether A. hydrophila from an aquaponic farm could form biofilms in aquaponic water and on materials used in these systems. A. hydrophila biofilm biomass and cell density in aquaponic water were evaluated by crystal violet staining and culture-based enumeration. Biofilm biomass and biofilm cell density were affected by the water source and A. hydrophila isolate (P < 0.05). A. hydrophila formed the most biomass from the beginning of deep-water culture (BDWC) water (OD570 0.202 ± 0.066) and the least from the end of deep-water culture (EDWC) water (OD570 0.140 ± 0.036; P < 0.05). Enumerated A. hydrophila from the biofilm varied among water sources; the fish tank water supported the greatest cell density (7.04 ± 0.71 log CFU/mL) while the EDWC supported the lowest cell density (6.76 ± 0.83 log CFU/mL). Biofilm formation was also evaluated on aquaponic materials such as nylon, polyvinyl chloride, polyethylene liner, bead filter, and foam. Biofilm formation on the liner had the greatest population (2.39 ± 0.022 log CFU/cm2), and the bead had the least (0.64 ± 0.039 log CFU/cm2; P < 0.05). Pathogenic organisms, such as A. hydrophila, may pose a greater risk to produce harvested from the BDWC and MDWC due to greater biofilm formation.

Isolation and genotypic characterization of extended-spectrum beta-lactamase-producing Escherichia coli O157:H7 and Aeromonas hydrophila from selected freshwater sources in Southwest Nigeria

The proliferation of antibiotic-resistant bacteria and antimicrobial resistance is a pressing public health challenge because of their possible transfer to humans via contact with polluted water sources. In this study, three freshwater resources were assessed for important physicochemical characteristics as well as heterotrophic and coliform bacteria and as potential reservoirs for extended-spectrum beta-lactamase (ESBL) strains. The physicochemical characteristics ranged from 7.0 to 8.3; 25 to 30 °C, 0.4 to 93 mg/L, 0.53 to 8.80 mg/L and 53 to 240 mg/L for pH, temperature, dissolved oxygen (DO), biological oxygen demand (BOD5) and total dissolved solids, respectively. The physicochemical characteristics mostly align with guidelines except for the DO and BOD5 in some instances. Seventy-six (76) Aeromonas hydrophila and 65 Escherichia coli O157: H7 isolates were identified by preliminary biochemical analysis and PCR from the three sites. Among these, A. hydrophila displayed higher frequencies of antimicrobial resistance, with all 76 (100%) isolates completely resistant to cefuroxime and cefotaxime and with MARI ≥ 0.61. The test isolates showed more than 80% resistance against five of the ten test antimicrobials, with resistance against cefixime, a cephalosporin antibiotic being the highest at 95% (134/141). The frequency of the detection of the resistance genes in the A. hydrophila isolates generally ranged between 0% (blaSHV) and 26.3% (blaCTX-M), while the frequency of detection among the E. coli O157:H7 isolates ranged between 4.6% (blaCTX-M) and 58.4% (blaTEM). Our findings indicate that the distribution of antibiotic-resistant bacteria with diverse ESBL-producing capabilities and virulence genes in freshwater sources potentially threatens public health and the environment.

Mechanisms of low susceptibility to the disinfectant benzalkonium chloride in a multidrug-resistant environmental isolate of Aeromonas hydrophila.

Excessive discharge of quaternary ammonium disinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, from a wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three different concentrations of BAC and characterized mechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 different sequenced A. hydrophila strains, consists of approximately 4.6 Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966. We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins. In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations. Other genes related to membrane fatty acid metabolism and redox metabolic reactions also showed an altered expression. Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand how bacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments.

Isolation and characterization of Aeromonas hydrophila lytic phage, and evaluation of a phage cocktail against A. hydrophila contamination in fish fillet

Besides being a finfish pathogen, Aeromonas hydrophila is also considered an important human enteropathogen. Phage biocontrol is increasingly being accepted as a natural and green technology for the control of bacterial pathogens as well as for minimizing the emergence/spread of antimicrobial resistance. In the present study, an A. hydrophila lytic phage (named D6) was isolated, characterized and subjected to whole-genome sequencing. During host range analysis, phage D6 belonging to order Caudovirales and family Myoviridae was able to lyse 95% of A. hydrophila isolates. The adsorption rate, latent period and burst size of phage D6 were estimated to be ∼95%, ∼60 min and 695 ± 44 plaque-forming unit (PFU)/host cell, respectively. The complete, circularly permuted 259,831 bp long phage genome consisted of 267 open reading frames (ORFs) and 3 tRNA genes. The genome of D6 also contained several genes essential for the phage life cycle. During in vitro host cell lysis test, phage D6 significantly inhibited the bacterial growth until 10 h at all the tested multiplicity of infection (MOI) values. A two-phage cocktail of D6 and previously isolated phage CF7 reduced the A. hydrophila counts by >95% in fish fillets at room temperature (25 °C) as well as at 4 °C. The comparison of phage counts at the start and end of the experiments also confirmed that both phage D6 and CF7 were able to propagate within the A. hydrophila host on the fillet matrix. Besides, phage counts on host-free fillets were stable throughout the experiments. The broad range phage cocktail from the present study could be used to ensure the safety of fishery products against A. hydrophila contamination.

Emergence of colistin resistance and characterization of antimicrobial resistance and virulence factors of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from hybrid red tilapia cage culture.

Tilapia is a primary aquaculture fish in Thailand, but little is known about the occurrence of antimicrobial resistance (AMR) in Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae colonizing healthy tilapia intended for human consumption and the co-occurrence of these AMR bacteria in the cultivation water. This study determined the phenotype and genotype of AMR, extended-spectrum β-lactamase (ESBL) production, and virulence factors of A. hydrophila, Salmonella spp., and V. cholerae isolated from hybrid red tilapia and cultivation water in Thailand. Standard culture methods such as USFDA's BAM or ISO procedures were used for the original isolation, with all isolates confirmed by biochemical tests, serotyping, and species-specific gene detection based on PCR. A total of 278 isolates consisting of 15 A. hydrophila, 188 Salmonella spp., and 75 V. cholerae isolates were retrieved from a previous study. All isolates of A. hydrophila and Salmonella isolates were resistance to at least one antimicrobial, with 26.7% and 72.3% of the isolates being multidrug resistant (MDR), respectively. All A. hydrophila isolates were resistant to ampicillin (100%), followed by oxytetracycline (26.7%), tetracycline (26.7%), trimethoprim (26.7%), and oxolinic acid (20.0%). The predominant resistance genes in A. hydrophila were mcr-3 (20.0%), followed by 13.3% of isolates having floR, qnrS, sul1, sul2, and dfrA1. Salmonella isolates also exhibited a high prevalence of resistance to ampicillin (79.3%), oxolinic acid (75.5%), oxytetracycline (71.8%), chloramphenicol (62.8%), and florfenicol (55.3%). The most common resistance genes in these Salmonella isolates were qnrS (65.4%), tetA (64.9%), bla TEM (63.8%), and floR (55.9%). All V. cholerae isolates were susceptible to all antimicrobials tested, while the most common resistance gene was sul1 (12.0%). One isolate of A. hydrophila was positive for int1, while all isolates of Salmonella and V. cholerae isolates were negative for integrons and int SXT. None of the bacterial isolates in this study were producing ESBL. The occurrence of mcr-3 (20.0%) in these isolates from tilapia aquaculture may signify a serious occupational and consumer health risk given that colistin is a last resort antimicrobial for treatment of Gram-negative bacteria infections. Findings from this study on AMR bacteria in hybrid red tilapia suggest that aquaculture as practiced in Thailand can select for ubiquitous AMR pathogens, mobile genetic elements, and an emerging reservoir of mcr and colistin-resistant bacteria. Resistant and pathogenic bacteria, such as resistance to ampicillin and tetracycline, or MDR Salmonella circulating in aquaculture, together highlight the public health concerns and foodborne risks of zoonotic pathogens in humans from cultured freshwater fish.

A Global Survey of Hypervirulent Aeromonas hydrophila (vAh) Identified vAh Strains in the Lower Mekong River Basin and Diverse Opportunistic Pathogens from Farmed Fish and Other Environmental Sources.

Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthalmus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo-inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.