Co-infection of Lactococcus garvieae and Aeromonas hydrophila in cultured Nile Tilapia in Kerala, India.

Co-infection of Lactococcus garvieae and Aeromonas hydrophila, has been confirmed from diseased Nile Tilapia (Oreochromis niloticus), Chithralada strain cultured in a freshwater rearing pond of Alappuzha district of Kerala, India. The aetiological agents behind the disease outbreak were bacteriologically proven and confirmed by 16SrRNA sequencing and phylogenetic analysis. PCR detection of the virulent genes, showed existence of adhesin and hemolysin in L. garvieae and aerolysin in A. hydrophila strain obtained. To fulfil Koch's postulates, challenge experiments were conducted and median lethal dose (LD50) of L. garvieae and A. hydrophila was calculated as 1 × 105.91 CFU per mL and 1 × 105.2 CFU per mL respectively. Histopathologically, eyes, spleen, and kidney were the predominantly infected organs by L. garvieae and A. hydrophila. Out of the 13 antibiotics tested to check antibiotic susceptibility, L. garvieae showed resistance to almost 7 antibiotics tested, with a resistance to Ciprofloxacin while A. hydrophila was found resistant to Streptomycin and Erythromycin. Understanding the complex interaction between Gram-positive and Gram-negative bacteria in the disease process and pathogenesis in fish host will contribute to efficient treatment strategies. As a preliminary investigation into this complex interaction, the present study is aimed at phenotypic and genotypic characterization, pathogenicity evaluation, and antibiotic susceptibility of the co-infecting pathogens in a diseased sample of freshwater-farmed Nile tilapia.

Genotype diversity and antibiotic resistance risk in Aeromonas hydrophila in Sichuan, China.

Sichuan is a significant aquaculture province in China, with a total aquaculture output of 1.72 × 106 tons in 2022. One of the most significant microorganisms hurting the Sichuan aquaculture is Aeromonas hydrophila, whose genotype and antibiotic resistance are yet unknown. This study isolated a total of 64 strains of A. hydrophila from various regions during September 2019 to June 2021 within Sichuan province, China. The technique of Multi-Locus Sequence Typing (MLST) was used for the purpose of molecular typing. Meanwhile, identification of antibiotic resistance phenotype and antibiotic resistance gene was performed. The findings of the study revealed that 64 isolates exhibited 29 sequence types (ST) throughout different regions in Sichuan, with 25 of these ST types being newly identified. Notably, the ST251 emerged as the predominant sequence type responsible for the pandemic. The resistance rate of isolated strains to roxithromycin was as high as 98.3%, followed by co-trimoxazole (87.5%), sulfafurazole (87.5%), imipenem (80%), amoxicillin (60%), and clindamycin (57.8%). Fifteen strains of A. hydrophila exhibited resistance to medicines across a minimum of three categories, suggesting the development of multidrug resistance in these isolates. A total of 63 ARGs were detected from the isolates, which mediated a range of antibiotic resistance mechanisms, with deactivation and efflux potentially serving as the primary mechanisms of antibiotic resistance. This study revealed the diversity of A. hydrophila genotypes and the risk of antibiotic resistance in Sichuan, providing reference for scientific and effective control of A. hydrophila infection.

Molecular characterization of Aeromonas hydrophila isolates from diseased fishes in district Kasur, Punjab, Pakistan / Caracterização molecular de isolados de Aeromonas hydrophila de peixes doentes no distrito de Kasur, Punjab, Paquistão

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.

Resumo O Paquistão é um país agrícola, onde a pesca desempenha um papel muito importante para o desenvolvimento econômico. Diferentes doenças são prevalentes em peixes do Paquistão, mas as informações relacionadas aos agentes causadores não são bem conhecidas. Tendo em vista a importância dos patógenos bacterianos como agentes causadores de múltiplas doenças em peixes, o presente estudo foi conduzido para identificação, caracterização e análise de genes de virulência de isolados de Aeromonas spp. de peixes doentes. Foram coletadas 50 amostras de peixes com múltiplas indicações clínicas em diferentes fazendas do distrito de Kasur, Punjab, Paquistão. Para isolar Aeromonas spp., as amostras foram enriquecidas e inoculadas em meio de isolamento. Os isolados foram identificados e caracterizados por diferentes testes bioquímicos, kit Analytical Profile Index (API) 20E, e ensaios de reação em cadeia da polimerase (PCR). Todos os isolados foram selecionados para três genes de virulência putativos, incluindo aerolisina (aer), hemolisina (hyl) e enterotoxina citotônica termolábil (alt). Sete isolados de Aeromonas hydrophila foram recuperados e identificados com base no API 20E. Esses isolados foram posteriormente confirmados como A. hydrophila de acordo com ensaios de PCR. Três isolados indicaram a presença de genes de virulência (alt e hyl), enquanto o gene aerolisina (aer) não esteve presente em nenhum dos isolados de A. hydrophila. O presente estudo confirmou A. hydrophila como o agente causador da síndrome ulcerativa epizoótica e septicemia móvel por Aeromonas em fazendas de peixes, no distrito de Kasur, Punjab, Paquistão. Além disso, a detecção de dois genes de virulência (alt e hyl) em isolados de A. hydrophila é uma ameaça para os consumidores de peixes da área de estudo.

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

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.

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.

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures.

Three different polyhydroxyalkanoate (PHA) synthase genes (Ralstonia eutropha H16, Aeromonas sp. TSM81 or Aeromonas hydrophila ATCC7966 phaC) were introduced into the chromosome of two Pseudomonas strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHAMCL) producer (Pseudomonas sp. LFM046) and a UV-induced mutant strain unable to produce PHA (Pseudomonas sp. LFM461). We reported for the first time the insertion of a chromosomal copy of phaC using the transposon system mini-Tn7. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-co-3HAMCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring A. hydrophila phaC produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of Pseudomonas sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HAMCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.

Hemerythrin is required for Aeromonas hydraphlia to survive in the macrophages of Anguilla japonica.

Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.

FlgN plays important roles in the adhesion of Aeromonas hydrophila to host mucus.

Adhesion to the host mucus is a crucial step in the early infection stage of pathogenic bacteria. To investigate the mechanisms of the adhesion of Aeromonas hydrophila to its host mucus, a mutant library was constructed using the mini-Tn10 transposon mutagenesis system. Of 276 individual colonies, the mutant strain with the most attenuated adhesion ability in this study was screened out and designated A77. Molecular analysis showed that a 414-bp sequence flanking mini-Tn10 in A77 had the highest identity (97%) with the bacterial flagellar protein gene flgN. A complemented strain flgN+ was constructed and the biological characteristics of the wild-type, mutant A77, and complemented flgN+ strains were investigated. The results showed that the decreased abilities of motility, adhesion to mucus, and biofilm formation in the mutant strain were partially recovered in the complemented flgN+ strain, which suggested that flgN plays an important role in the adhesion of A. hydrophila to its host.

Role of MshQ in MSHA pili biosynthesis and biofilm formation of Aeromonas hydrophila.

Biofilm formation of pathogen bacterium is currently one of the most widely studied topics; however, little is known regarding pathogen bacteria biofilms in aquaculture. Aeromonas hydrophila is a representative species of the genus Aeromonas, which has been recognized as a common pathogen, is associated with many diseases in aquatic animals, and causes significant mortality. The objectives of this study are i) to confirm that A. hydrophila can form biofilms on abiotic substrates and construct a biofilm growth curve for this bacterium; ii) to identify the genes that play crucial roles in A. hydrophila biofilm formation. The biofilm growth curve of A. hydrophila was constructed using a crystal violet assay, which showed that biofilm formation for this bacterium is a dynamic process. Next, a mutant library of pathogenic A. hydrophila B11 was constructed using the mini-Tn10 transposon mutagenesis system. A total of 861 mutants were screened, and 5 mutants were stably deficient in biofilm formation. Molecular analysis of the mutant B112 revealed that the open reading frame that encodes the protein MshQ was disrupted. Comparison of biological characteristics including growth, motility, and adhesion between the mutant B112 and the wild-type strain B11 suggested that MshQ is necessary for mannose-sensitive hemagglutinin pilus biosynthesis of A. hydrophila, and that these pili play crucial roles in A.hydrophila adherence to a solid surface during the early stages of biofilm formation.

Inability of some Aeromonas hydrophila strains to act as recipients of plasmid pRAS1 in conjugal transfer experiments.

Plasmids belonging to the IncU incompatibility group are mobile genetic elements isolated frequently from Aeromonas spp. These plasmids share structural and functional characteristics and often carry Class-1 integrons bearing antibiotic resistance genes. In this work the ability of two IncU plasmids, pAr-32 and pRAS1 to establish in different A. hydrophila strains after conjugal transfer was studied. In vitro transfer frequencies on solid surface ranged from 10(-1) to 10(-6) for pAr-32 and from 10(-3) to 10(-5) for pRAS1. While carrying out these experiments we detected four strains unable to acquire plasmid pRAS1, indicating that the genetic background of recipients affects the establishment of the plasmid. We explored the possible reasons why these strains failed to yield transconjugants after mating experiments using A. salmonicida 718 as a donor. Factors included donor cell recognition, incompatibility, surface exclusion and restriction of incoming DNA. We found that none of these factors could explain the refractivity of non-receptive A. hydrophila strains to yield transconjugants. Although we do not know the reasons of this refractivity, we may speculate that these isolates lack a product necessary to replicate or stabilize plasmid pRAS1. Alternatively, these strains could contain a product that impedes plasmid establishment.

Presence of blaTEM-116 gene in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei from Brazil

It is known that Aeromonas spp. possess different chromosomal â-lactamase genes. Presence and phenotypic expression of blaTEM, blaSHV, and blaCTX-M ESBL-encoding genes were investigated in environmental water isolates of Aeromonas hydrophila and Aeromonas jandaei. Presence of blaSHV and blaCTX-M genes was not observed, and blaTEM gene was verified in 91 percent of the isolates. Sequencing of 10 fragments showed the occurrence of blaTEM-116.

Detection of type III secretion system genes in Aeromonas hydrophila and their relationship with virulence in Nile tilapia.

The goals of this study were to develop a PCR technique to detect ascV and aopB genes from the type III secretion system (T3SS), to evaluate the frequency of these genes in Aeromonas hydrophila strains isolated from diseased fish and from aquaculture environments, and to determine the relationship between the presence of these genes and virulence of A. hydrophila in Nile tilapia. The PCR assay developed here successfully detected the target genes, showing three different profiles for the strains ascV+/aopB+, ascV+/aopB-, and ascV-/aopB-. A higher frequency of ascV+/aopB+ was verified in isolates from diseased fish compared to those from aquaculture environments (P<0.05). Among 64 isolates from diseased fish, ascV+/aopB+ (62.5%) was the most frequent profile (P<0.05) and caused more intensive mortality rates. Environmental strains containing the ascV+/aopB+ profile were less virulent than isolates from clinical cases. These results suggest that the presence of a functional T3SS probably increases the virulence of A. hydrophila. The PCR technique was shown to be a specific and efficient tool for detection of T3SS, and this technique can be used for virulence typing of A. hydrophila isolates.

In vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and Aeromonas hydrophila in artificially infected Cyprinus carpio L.

This study investigated the possible in vivo transfer of plasmid pRAS1 between Aeromonas salmonicida and A. hydrophila inhabiting two different organs of Cyprinus carpio L. To distinguish transconjugants from naturally occurring antibiotic resistant bacteria, twelve luminescent transposon-tagged A. hydrophila strains using mini Tn5luxCDABEKm2 transposon were generated. In conjugal transfer experiments, fish were conditioned with the donor bacteria and subsequently immersed in water containing the recipient strain. Bacteria were recovered from gills and intestines and isolated by growth on selective plates. Transconjugants were identified by their resistance to the pRAS1 encoded antimicrobials and by light emission. In vivo transfer frequencies ranged between 10(-3) and 10(-6) and were somewhat lower in intestines, compared to gills. Transfer frequencies were also smaller relative to those obtained in vitro. The minimal amount of donor and recipient bacteria needed to yield detectable transconjugants in vivo was 1 x 10(4) CFU mL(-1). Implications of this plasmid transfer in natural settings and its possible consequences to human health are discussed.

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei.

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hlyA/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hlyA/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.

Aeromonas hydrophila clinical and environmental ecotypes as revealed by genetic diversity and virulence genes.

Aeromonas hydrophila strains recovered from clinical samples and ambient sources were phenotypically and genetically identified. In addition, the distribution of putative virulence factors was assayed. To determine the genetic diversity of these strains, random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR markers were used. The discriminatory ability of the techniques, using Simpson's index, was 0.96 for both methods. The most consistent dendrogram was obtained when RAPD and ERIC data were combined. The genetic diversity revealed a high intra-specific genetic diversity (h=0.364+/-0.024 and I=0.538+/-0.030). The strains showed a tendency to cluster according to their origin of isolation (best-cut test 0.80 and bootstrap values >50%). The present study demonstrates and quantifies the high intra-specific diversity within this species and reveals a clear differentiation of strains according to their ecological origin. The distribution of virulence-related genes confirm that A. hydrophila is a genetically heterogeneous species that harbour ecotypes which have different pathogenic potential to human and other animals.

Susceptibility to antimicrobial agents and plasmid carrying in Aeromonas hydrophila isolated from two estuarine systems.

Susceptibility to various antimicrobial agents and the presence of plasmids was investigated in eleven strains of Aeromonas hydrophila isolated from samples of sea water and these strains isolated from Aulacomya ater. Transference of resistance to Escherichia coli was attempted by conjugation and transformation experiments. The strains showed multiple resistance toward beta-lactam antibiotics and susceptibility to other antimicrobial agents. Five strains harboured plasmids with molecular weights below 5.7 MD. It was not possible to relate the resistance of the strains with the presence of their plasmids.