Sudden death caused by Aeromonas hydrophila in a Crab-eating Macaque (Macaca fascicularis) associated with captivity stress.

Aeromonas hydrophila is a facultative anaerobic gram-negative bacterium regarded as an opportunistic pathogen in animals. A 17-year-old female crab-eating macaque (Macaca fascicularis) died after experiencing anorexia and depression for several days. The carcass was severely emaciated, and the sternum was exposed under subcutaneous lesions in the thorax. Many abnormal pathological lesions were found, including tracheal inflammation, pulmonary inflammatory emphysema, yellowish discoloration of the liver, enlargement of the gall bladder, necrosis of the heart, congested bilateral kidneys, and enlargement of the adrenal glands. The stomach was empty, mucosal ulcerations were found, and the duodenum was congested. Giemsa staining revealed rod-shaped organisms in the whole blood smear and major organs, which were identified as A. hydrophila. The animal had experienced stress, and decreased immune system function possibly contributed to the infection.

Microbial Water Quality Assessment of Private Wells Using 16S rRNA Gene Amplicon Sequencing with a Nanopore Sequencer.

Private wells are used daily worldwide as convenient household water sources. In Japan, where water supply coverage is high, well water is occasionally used for non-potable purposes, such as irrigation and watering. Currently, the main microbiological test of well water is designed to detect Escherichia coli, which is an indicator of fecal contamination, using culture methods. Water use such as watering generates bioaerosols, which may cause airborne infection. However, many causative bacteria of aerosol-derived infections, such as Legionella spp., are difficult to detect using culture methods. Thus, more comprehensive modern assessment is desirable for securing the microbiological quality of well water. Here, the bacterial community structure of five private wells located in different environments was examined using the rapid and portable MinION sequencer, which enabled us to identify bacteria to the species level based on full-length 16S ribosomal RNA (rRNA) gene sequences. The results revealed the differences in the bacterial community structures of water samples from the five wells and detected Legionella pneumophila and Aeromonas hydrophila as new candidate microbial indicators. The comprehensive analysis method used in this study successfully detected bacteria causing opportunistic infections, which are difficult to detect by conventional methods. This approach is expected to be routinely applied in the future as a highly accurate method for assessing the microbiological quality of private well water.

Epidemiological study of ticks harbouring Aeromonas hydrophila in areas endemic and non-endemic to Japanese-spotted fever.

OBJECTIVES: Aeromonas spp. often cause life-threatening diseases, including necrotizing fasciitis, which may lead to septic shock and ultimately death. Aeromonas infections are believed to be transmitted via minor wounds or the consumption of fresh fish. However, after the detection of Aeromonas hydrophila in ticks in areas endemic to Japanese-spotted fever (JSF), a novel transmission route of A. hydrophila (i.e., via tick bites) has been proposed. We investigated the prevalence of A. hydrophila in ticks in areas endemic and not endemic to JSF in the Mie Prefecture, Japan. METHODS: We collected ticks from endemic and nonendemic areas in summer and winter and assessed them for presence of A. hydrophila using polymerase chain reaction. RESULTS: Six A. hydrophila isolates were obtained from 95 ticks in endemic areas, whereas one A. hydrophila isolate was obtained from 142 ticks in non-endemic areas, in summer. All ticks that harboured A. hydrophila were Haemaphysalis longicornis (H.L); these ticks were almost at the larval stage and also carried Rickettsia spp. in the endemic area. In contrast, 51 and 41 ticks in the endemic and non-endemic areas were captured in winter, respectively; A. hydrophila was not detected in these. CONCLUSIONS: This study revealed the prevalence of tick-borne A. hydrophila. Therefore, the risk of transmission of A. hydrophila via a tick bite should be considered in the following conditions: areas abundant in H. L. harbouring Rickettsia spp., in areas endemic for JSF, presence of ticks in the larval stage and during the summer season.

Isolation and characterization of two virulent Aeromonads associated with haemorrhagic septicaemia and tail-rot disease in farmed climbing perch Anabas testudineus.

Diseased Anabas testudineus exhibiting signs of tail-rot and ulcerations on body were collected from a fish farm in Assam, India during the winter season (November 2018 to January 2019). Swabs from the infected body parts were streaked on sterilized nutrient agar. Two dominant bacterial colonies were obtained, which were then isolated and labelled as AM-31 and AM-05. Standard biochemical characterisation and 16S rRNA and rpoB gene sequencing identified AM-31 isolate as Aeromonas hydrophila and AM-05 as Aeromonas jandaei. Symptoms similar to that of natural infection were observed on re-infecting both bacteria to disease-free A. testudineus, which confirmed their virulence. LC50 was determined at 1.3 × 104 (A. hydrophila) and 2.5 × 104 (A. jandaei) CFU per fish in intraperitoneal injection. Further, PCR amplification of specific genes responsible for virulence (aerolysin and enterotoxin) confirmed pathogenicity of both bacteria. Histopathology of kidney and liver in the experimentally-infected fishes revealed haemorrhage, tubular degeneration and vacuolation. Antibiotic profiles were also assessed for both bacteria. To the best of our knowledge, the present work is a first report on the mortality of farmed climbing perch naturally-infected by A. hydrophila as well as A. jandaei, with no records of pathogenicity of the latter in this fish.

Morphology, phylogenetics and pathology of "red sore disease" (coinfection by Epistylis cf. wuhanensis and Aeromonas hydrophila) on sportfishes from reservoirs in the South-Eastern United States.

The aetiological agents of red sore disease (RSD) reportedly comprise a taxonomically ambiguous stalked ciliate (a species of Epistylis) and Aeromonas hydrophila. The taxonomic identity of each pathogen remains provisional: using supra-specific morphological features for the ciliate and culture-based methods that cannot delineate bacterial strain. On 7 and 9 November 2017 and 28 May 2020, biologists and anglers reported a local epizootic (Hiwassee and Chattahoochee river basins; Georgia) wherein some moribund fish presented RSD-like lesions. The ciliates were assigned to Epistylis by morphology. The ciliate is regarded as Epistylis cf wuhanensis, as nucleotide sequences from its small subunit ribosomal DNA were identical to those of Epistylis wuhanensis. The bacterium was identified as Aeromonas hydrophila by phenotypic markers and nucleotide sequences from the DNA gyrase subunit B; our sequences comprised 3 strains and phylogenetically were recovered sister to strains of Eurasian origin. Histological sections of lesions revealed effacement or partial deterioration of the epithelium covering scales, scale loss, haemorrhaging, necrosis, oedema, and extensive inflammatory infiltrate in the dermis. This is the first nucleotide sequence information for the symbionts implicated in RSD.

Broad-Spectrum Robust Direct Bactericidal Activity of Fish IFNφ1 Reveals an Antimicrobial Peptide-like Function for Type I IFNs in Vertebrates.

Type I IFNs (IFN-Is) play pivotal roles in host defense against viral infections but remain enigmatic against bacterial pathogens. In this study, we recombinantly expressed and purified intact grass carp (Ctenopharyngodon idella) IFNφ1 (gcIFNφ1), a teleost IFN-I. gcIFNφ1 widely powerfully directly kills both Gram-negative and Gram-positive bacteria in a dose-dependent manner. gcIFNφ1 binds to LPS or peptidoglycan and provokes bacterial membrane depolarization and disruption, resulting in bacterial death. Furthermore, gcIFNφ1 can efficiently protect zebrafish against Aeromonas hydrophila infection and significantly reduce the bacterial loads in tissues by an infection model. In addition, we wonder whether antibacterial IFN-I members exist in other vertebrates. The amino acid compositions of representative IFN-Is with strong positive charges from Pisces, Amphibia, reptiles, Aves, and Mammalia demonstrate high similarities with those of 2237 reported cationic antimicrobial peptides in antimicrobial peptide database. Recombinant intact representative IFN-I members from the nonmammalian sect exhibit potent broad-spectrum robust bactericidal activity through bacterial membrane depolarization; in contrast, the bactericidal activity is very weak from mammalian IFN-Is. The findings display a broad-spectrum potent direct antimicrobial function for IFN-Is, to our knowledge previously unknown. The results highlight that IFN-Is are important and robust in host defense against bacterial pathogens, and unify direct antibacterial and indirect antiviral bifunction in nonmammalian jawed vertebrates.

Simultaneous detection of multiple bacterial and viral aquatic pathogens using a fluorogenic loop-mediated isothermal amplification-based dual-sample microfluidic chip.

Rapid and user-friendly diagnostic tests are necessary for early diagnosis and immediate detection of diseases, particularly for on-site screening of pathogenic microorganisms in aquaculture. In this study, we developed a dual-sample microfluidic chip integrated with a real-time fluorogenic loop-mediated isothermal amplification assay (dual-sample on-chip LAMP) to simultaneously detect 10 pathogenic microorganisms, that is Aeromonas hydrophila, Edwardsiella tarda, Vibrio harveyi, V. alginolyticus, V. anguillarum, V. parahaemolyticus, V. vulnificus, infectious hypodermal and haematopoietic necrosis virus, infectious spleen and kidney necrosis virus, and white spot syndrome virus. This on-chip LAMP provided a nearly automated protocol that can analyse two samples simultaneously, and the tests achieved limits of detection (LOD) ranging from 100 to 10-1  pg/µl for genomic DNA of tested bacteria and 10-4 to 10-5  pg/µl for recombinant plasmid DNA of tested viruses, with run times averaging less than 30 min. The coefficient of variation for the time-to-positive value was less than 10%, reflecting a robust reproducibility. The clinical sensitivity and specificity were 93.52% and 85.53%, respectively, compared to conventional microbiological or clinical methods. The on-chip LAMP assay provides an effective dual-sample and multiple pathogen analysis, and thus would be applicable to on-site detection and routine monitoring of multiple pathogens in aquaculture.

Development of a real-time recombinase polymerase amplification assay for rapid detection of Aeromonas hydrophila.

Aeromonas hydrophila is ubiquitous in the aquaculture industry and a constant cause of severe disease and economic losses. The early diagnosis of these infections is crucial for disease surveillance and prevention. We developed a real-time recombinase polymerase amplification (real-time RPA) assay for detection of A. hydrophila using the haemolysin gene. The assay was performed at 37°C for 20 min and was highly specific with no cross-reaction with other fish pathogens or with other Aeromonas species. The assay detection limit was 102 copies of the Aeromonas hydrophila per reaction. Compared with traditional culture-based method or real-time PCR, the diagnostic sensitivity and specificity of the real-time RPA were 73.7 and 100%, as well as 64.7 and 93%. Our newly developed real-time RPA was specific and sensitive and can be used in large-scale and point-of-care field investigations of A. hydrophila infections to enable earlier diagnoses.

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.

Virulence-Associated Genes and Antimicrobial Resistance of Aeromonas hydrophila Isolates from Animal, Food, and Human Sources in Brazil.

Aeromonads are natural inhabitants of aquatic environments and may be associated with various human or animal diseases. Its pathogenicity is complex and multifactorial and is associated with many virulence factors. In this study, 110 selected Aeromonas hydrophila isolates isolated from food, animals, and human clinical material from 2010 to 2015 were analyzed. Antimicrobial susceptibility testing was performed by the disk diffusion method, and polymerase chain reaction was conducted to investigate the virulence genes hemolysin (hlyA), cytotoxic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), aerolysin (aerA), and DNase-nuclease (exu). At least 92.7% of the isolates had one of the investigated virulence genes. Twenty different virulence profiles among the isolates were recognized, and the five investigated virulence genes were observed in four isolates. Human source isolates showed greater diversity than food and animal sources. Antimicrobial resistance was observed in 46.4% of the isolates, and multidrug resistance was detected in 3.6% of the isolates. Among the 120 isolates, 45% were resistant to cefoxitin; 23.5% to nalidixic acid; 16.6% to tetracycline; 13.7% to cefotaxime and imipenem; 11.8% to ceftazidime; 5.9% to amikacin, gentamicin, and sulfamethoxazole-trimethoprim; and 3.9% to ciprofloxacin and nitrofurantoin. Overall, the findings of our study indicated the presence of virulence genes and that antimicrobial resistance in A. hydrophila isolates in this study is compatible with potentially pathogenic bacteria. This information will allow us to recognize the potential risk through circulating isolates in animal health and public health and the spread through the food chain offering subsidies for appropriate sanitary actions.

Comparative transcriptome and phenotype analysis revealed the role and mechanism of ompR in the virulence of fish pathogenic Aeromonas hydrophila.

Aeromonas hydrophila B11 strain was isolated from diseased Anguilla japonica, which had caused severe gill ulcers in farmed eel, causing huge economic losses. EnvZ-OmpR is a model two-component system in the bacteria and is widely used in the research of signal transduction and gene transcription regulation. In this study, the ompR of A. hydrophila B11 strain was first silenced by RNAi technology. The role of ompR in the pathogenicity of A. hydrophila B11 was investigated by analyzing both the bacterial comparative transcriptome and phenotype. The qRT-PCR results showed that the expression of ompR in the ompR-RNAi strain decreased by 97% compared with the wild-type strain. The virulence test showed that after inhibition of the ompR expression, the LD50 of A. hydrophila B11 decreased by an order of magnitude, suggesting that ompR is involved in the regulation of bacterial virulence. Comparative transcriptome analysis showed that the expression of ompR can directly regulate the expression of several important virulence-related genes, such as the bacterial type II secretion system; moreover, ompR expression also regulates the expression of multiple genes related to bacterial chemotaxis, motility, adhesion, and biofilm formation. Further studies on the phenotype of A. hydrophila B11 and ompR-RNAi also confirmed that the downregulation of ompR expression can decrease bacterial chemotaxis, adhesion, and biofilm formation.

Effect of curcumin on the expression of ahyI/R quorum sensing genes and some associated phenotypes in pathogenic Aeromonas hydrophila fish isolates.

Quorum sensing (QS) in Aeromonas hydrophila is mainly based on the modulation of ahyI/R genes that regulates bacterial virulence determinant phenotypes. The use of QS inhibitors would be of particular interest in inhibiting bacterial pathogenicity and infections. In this study, we aimed to determine the effect of curcumin, a natural component of Curcuma longa, on the expression of QS regulating genes, ahyI and ahyR, as well as some QS regulated virulence characteristics in pathogenic fish isolated A. hydrophila strains. The minimum inhibitory concentration (MIC) of curcumin against bacteria was determined using the broth micro-dilution method and the expression of quorum sensing genes ahyI and ahyR among the bacteria treated with curcumin was determined using quantitative polymerase chain reaction (qPCR). Also, the effect of curcumin on some QS associated traits, including biofilm formation, swarming and swimming motility, proteolytic potential, and bacterial hemolytic activity was investigated. According to the results, curcumin, at a concentration of 32 µg/mL, significantly reduced the expression of both ahyI and ahyR genes among bacterial strains up to 64.2 and 91.0%, respectively. Moreover, curcumin efficiently inhibited bacterial biofilm formation, swimming, and swarming motility. Also, bacterial proteolytic activity was slightly reduced, while hemolytic activity was not significantly affected. This study demonstrated the use of curcumin to attenuate ahyI/R QS genes and several QS associated phenotypes in A. hydrophila. These findings indicate the therapeutic potential of curcumin as an anti-QS agent, to be used against A. hydrophila pathogenesis in aquaculture.

Surgical site infection caused by Aeromonas hydrophila presenting as necrotizing soft tissue infection after esophagectomy.

Several virulence factors of Aeromonas such as hemolysin, proteases and lipases have been characterized. The relationship between these virulence factors and disease remains unclear. A 71-year-old man underwent thoracoscopic esophagectomy, lymph node dissection and Roux-en-Y reconstruction for esophageal cancer. On postoperative day 1, redness around the wound on the thoracic abdominal wall gradually enlarged and necrosis became apparent with septic shock. Necrotizing soft tissue infection was suspected and emergency surgical debridement was performed. Blood and wound cultures were positive for Aeromonas hydrophila. The strain was found to have hemolytic activity, proteolytic activity and extremely high elastolytic activity. In addition, the strain actively produced elastolytic metalloprotease, which may contribute to extensive tissue necrosis.

First data on microflora of loggerhead sea turtle (Caretta caretta) nests from the coastlines of Sicily.

Caretta caretta is threatened by many dangers in the Mediterranean basin, but most are human-related. The purposes of this research were: (i) to investigate microflora in samples from six loggerhead sea turtle nests located on the Sicilian coast and (ii) to understand microbial diversity associated with nests, with particular attention to bacteria and fungi involved in failed hatchings. During the 2016 and 2018 summers, 456 eggs and seven dead hatchling from six nests were collected. We performed bacteriological and mycological analyses on 88 egg samples and seven dead hatchlings, allowing us to isolate: Fusarium spp. (80.6%), Aeromonas hydrophila (55.6%), Aspergillus spp. (27.2%) and Citrobacter freundii (9%). Two Fusarium species were identified by microscopy and were confirmed by PCR and internal transcribed spacer sequencing. Statistical analyses showed significant differences between nests and the presence/absence of microflora, whereas no significant differences were observed between eggs and nests. This is the first report that catalogues microflora from C . caretta nests/eggs in the Mediterranean Sea and provides key information on potential pathogens that may affect hatching success. Moreover, our results suggest the need for wider investigations over extensive areas to identify other microflora, and to better understand hatching failures and mortality related to microbial contamination in this important turtle species.

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.

Virulence genes contributing to Aeromonas hydrophila pathogenicity in Oreochromis niloticus

Bacterial diseases are the main cause of high economic loss in aquaculture, particularly gram-negative bacteria. This study was conducted for the isolation and identification of Aeromonas and Pseudomonas spp. from diseased fish. Twenty-two Aeromonas and sixteen Pseudomonas isolates were recovered from diseased Nile tilapia (Oreochromis niloticus) raised in eight earthen ponds in Elhox, Metoubes, Kafrelsheikh, Egypt. The recovered isolates were further identified using PCR as 22 Aeromonas hydrophila, 11 Pseudomonas aeruginosa, and 5 Pseudomonas fluorescens isolates. The 22 A. hydrophila isolates were screened for the presence of four virulence genes. Sixteen of the isolates (72.72%) were positive for the aerolysin gene (aer); 4 (18.18%) harbored the cytotoxic enterotoxin gene (act); and 2 (9.09%) carried the hemolysin A gene (hylA) while the cytotonic heat-stable enterotoxin gene (ast) was absent from all the tested isolates. The pathogenicity test indicated the direct relationship between the mortality percentage and the genotype of the tested A. hydrophila isolates as the mortality rates were 63.3 and 73.3% for isolates with two virulence genes (aer+ & act+, and aer+ and hylA+, respectively), followed by 40, 53.3, and 56.6% for isolates with only one virulence gene (hylA, act, and aer, respectively) and 20% for isolates lacking virulence genes. Based on the sensitivity test, the multi-antibiotic resistance profiles were as follows: 90.9% of the A. hydrophila isolates were sensitive to florfenicol and doxycycline; then 68.18% were susceptible to oxytetracycline, norfloxacin, and ciprofloxacin; and 63.63% were susceptible to sulfamethoxazole-trimethoprim, while only 27.27 and 4.5% were sensitive to erythromycin and cephradine, respectively, and all the isolates were resistant to amoxicillin and ampicillin

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Molecular characteristics, pathogenicity and medication regimen of Aeromonas hydrophila isolated from common carp (Cyprinus carpio L.).

Aeromonas hydrophila causes disease in fish known as Motile Aeromonas Septicemia (MAS), also named as bacterial hemorrhagic septicemia. In this study, a pathogenic A. hydrophila strain was isolated from common carp Cyprinus carpio L., which were suffering from severe hemorrhagic septicemia. According to the phylogenetic analysis derived from 16S rDNA sequence, the isolate formed a single branch in the A. hydrophila group, named AhHN1. Artificial infection results indicated that AhHN1 showed strong pathogenicity in C. carpio and the LD50 was 1.38 × 106 CFU/fish, the clinical symptoms and pathological features of infected fish were similar to those observed in natural infections. The antimicrobial susceptibility testing revealed that AhHN1 resistance to more than 13 kinds of antimicrobial agents. However, the AhHN1 strain exhibited an extremely sensitivity to enrofloxacin, the in vitro activities of enrofloxacin were subsequently investigated and drug selection window (MSW) was 0.0016-0.0125 µg/ml. Pharmacokinetics data showed that plasma concentration of enrofloxacin was 0.0016, 0.0148 and 0.0282 µg/ml at 24 hr after orally administered with 2.5, 5 and 10 mg/kg enrofloxacin. Moreover, dosing once a day of 2.5, 5 and 10 mg/kg enrofloxacin, which the relative protection ratio (RPS) was amounted to 33.3, 66.7, and 83.3%, respectively. Therefore, 5 mg/kg enrofloxacin was considered to be the rational regimen for controlling AhHN1 infection in C. carpio in the countries where the use of enrofloxacin is permitted in aquaculture. The aim of this study was to establish a scientific medication regimen for the prevention and therapy of the mutidrug-resistant A. hydrophila infection.

Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan.

Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial-resistant (AMR) Aeromonas strains (A. hydrophila GSH8-2 and A. caviae GSH8M-1) carrying the carabapenemase blaKPC-2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8-2 and GSH8M-1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired blaKPC-2 -positive IncP-6-relative plasmids (pGSH8-2 and pGSH8M-1-2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to blaKPC-2 , pGSH8M-1-2 carries an IS26-mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities.

Roles of three TonB systems in the iron utilization and virulence of the Aeromonas hydrophila Chinese epidemic strain NJ-35.

The TonB system functions in iron transport and has been identified in certain Gram-negative bacteria. Recently, we reported three TonB systems in the Aeromonas hydrophila Chinese epidemic strain NJ-35, but the functions of these systems have not been thoroughly elucidated to date. In this study, we investigated the role of these TonB systems in A. hydrophila iron utilization and virulence. We found that tonB1 and tonB2 were preferentially transcribed in iron-chelated conditions, where gene expression levels were approximately 8- and 68-fold higher compared with iron-rich conditions, respectively; tonB3 was consistently transcribed at a low level under iron-repleted and iron-depleted conditions. Only the TonB2 system was required to utilize iron-binding proteins. The tonB123 mutant showed increased susceptibility to erythromycin and roxithromycin. In addition, all three tonB genes were involved in A. hydrophila virulence in zebrafish, and various phenotypes associated with environmental survival were changed with varying degrees in each tonB mutant. TonB2 plays a relatively major role in adhesion, motility, and biofilm formation, while TonB3 is more involved in the anti-phagocytosis of A. hydrophila. In each observed phenotype, no significant difference was found between the single- and double-deletion mutants, whereas the triple-deletion mutant exhibited the most serious defects, indicating that all three TonB systems of A. hydrophila coordinately complement one another. In conclusion, this study elucidates the importance of TonB in iron acquisition and virulence of A. hydrophila, which lays the foundation for future studies regarding the survival mechanisms of this bacterium in iron-restricted environments.

Predictive Modeling for the Growth of Aeromonas hydrophila on Lettuce as a Function of Combined Storage Temperature and Relative Humidity.

This study developed predictive growth models of Aeromonas hydrophila on lettuce as a function of combined storage temperature (15-35°C) and relative humidity (RH, 60-80%) using a polynomial equation. The primary model of specific growth rate, lag time, and maximum population density showed a good fit (R2 ≥ 0.95) with a Gompertz equation. A secondary model was obtained using a quadratic polynomial equation. The appropriateness of the secondary model was verified by mean square error (0.0001-0.8848), bias factor (Bf = 0.962-1.009), and accuracy factor (Af = 1.002-1.104). The newly developed secondary models for A. hydrophila could be incorporated into the tertiary modeling program to predict the growth of A. hydrophila as a function of combined temperature and RH. The developed model may be useful to predict potential A. hydrophila growth on lettuce, which is important for food safety purpose during the overall food chain of lettuce from farm to table. It could offer reliable and useful information of growth kinetics for the quantification of microbial risk assessment of A. hydrophila on lettuce.